Compositions and Methods for Kallikrein (KLKB1) Gene Editing

ABSTRACT

Compositions and methods for editing, e.g., introducing double-stranded breaks, within the KLKB1 gene are provided. Compositions and methods for treating subjects having hereditary angioedema (HAE), are provided.

This patent application is a continuation of International ApplicationNo. PCT/US2021/016730, filed Feb. 5, 2021, which claims priority to U.S.Provisional Patent Application No. 62/971,906, filed Feb. 7, 2020; U.S.Provisional Patent Application No. 62/981,965, filed Feb. 26, 2020; andU.S. Provisional Patent Application No. 63/019,076, filed May 1, 2020,the contents of each of which are incorporated herein by reference intheir entirety for all purposes.

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Sep. 1, 2022, isnamed “2022-09-01_01155-0031-00US_ST26” and is 593,149 bytes in size.

Hereditary angioedema (HAE) affects one in 50,000 people and contributesto 15,000 to 30,000 emergency room visits per year. HAE is a rareautosomal, dominantly inherited blood disorder characterized byrecurrent episodes of severe swelling (angioedema). The most commonareas of the body to develop swelling are the limbs, face, GI tract, andairway. Minor trauma or stress may trigger an attack but swelling oftenoccurs without a known trigger. Episodes involving the intestinal tractcause severe abdominal pain, nausea, and vomiting. Swelling in theairway can restrict breathing and lead to life-threatening obstructionof the airway or asphyxiation. Symptoms of HAE typically begin inchildhood and worsen during puberty. On average, untreated individualshave an attack every 1 to 2 weeks, and most episodes last for about 3 to4 days. There are three types of hereditary angioedema, called types I,II, and III, and the different types have similar signs and symptoms.

Hereditary angioedema stems from excess bradykinin in the bloodpromoting vascular permeability and episodes of swelling. Most patientswith HAE have a C1 inhibitor (also called C1 esterase inhibitor orC1-INH) protein deficiency. In the absence of C1-INH, bradykinin levelscan rise, initiate vascular leakage, and cause swelling attacks. Itsproduction is controlled via the kallikrein-kinin (contact) pathwaywhich is endogenously inhibited by C1-INH. Bradykinin peptide is formedwhen high-molecular weight kininogen (HMWK) is cleaved by plasmakallikrein (pKal), an activated form of the protein prekallikrein.Prekallikrein is encoded by KLKB1 and is also called KLKB1 protein.KLKB1 protein is produced in the liver and secreted into plasma where itcan be activated by factor XIIa. Once KLKB1 is activated, pKal canincrease bradykinin levels. An excess of bradykinin in the blood leadsto fluid leakage through the walls of blood vessels into body tissues.Excessive accumulation of fluids in body tissues causes the episodes ofswelling seen in individuals with HAE.

Several drugs targeting the kallikrein-kinin pathway have beendeveloped, including C1 esterase inhibitors (Berinert®, Cinryze®),recombinant C1-INH replacement therapy (rhC1INH; conestat alfa (Rhucin®,Ruconest®)), and bradykinin receptor antagonist (Icatibant, Firazyr®).Approaches using kallikrein or prekallikrein (KLKB1) inhibitors alsohave been developed (ecallantide, Kalbitor®; lanadelumab, Takhzyro™).

The present disclosure provides compositions and methods using theCRISPR/Cas system to knock out the KLKB1 gene, thereby reducing theproduction of prekallikrein (KLKB1), reducing kallikrein, and reducingbradykinin production in subjects with HAE.

Accordingly, the following embodiments are provided. In someembodiments, the present invention provides compositions and methodsusing a guide RNA with an RNA-guided DNA binding agent such as theCRISPR/Cas system to substantially reduce or knockout expression of theKLKB1 gene, thereby substantially reducing or eliminating the productionof bradykinin. The substantial reduction or elimination of theproduction of bradykinin through alteration of the KLKB1 gene can be along-term or permanent treatment.

The following embodiments are provided herein.

Embodiment A1 is a guide RNA comprising:

-   -   a. a guide sequence comprising at least 95%, 90%, or 85%        identical to a sequence selected from SEQ ID NOs: 15, 8, and 41;    -   b. a guide sequence comprising at least 17, 18, 19, or 20        contiguous nucleotides of a sequence selected from SEQ ID NOs:        15, 8, and 41; or    -   c. a guide sequence selected from SEQ ID NOs: 15, 8, and 41.

Embodiment A2 is the guide RNA of embodiment A1, further comprising thenucleotide sequence of SEQ ID NO: 202.

Embodiment A3 is the guide RNA of embodiment A1, wherein the guide RNAfurther comprises a nucleotide sequence selected from SEQ ID NO: 170,171, 172, and 173 wherein the sequence of SEQ ID NO: 170, 171, 172, or173 is 3′ of the guide sequence.

Embodiment A4 is the guide RNA of any one of embodiments A1-A3, whereinthe guide RNA further comprises a 3′ tail.

Embodiment A5 is the guide RNA of any one of embodiments A1-A4, whereinthe guide RNA comprises at least one modification.

Embodiment A6 is the guide RNA of embodiment A5, wherein themodification comprises a 5′ end modification.

Embodiment A7 is the guide RNA of embodiment A5 or A6, wherein themodification comprises a 3′ end modification.

Embodiment A8 is the guide RNA of any one of embodiments A1-A7, whereinthe guide RNA comprises a modification in a hairpin region.

Embodiment A9 is the guide RNA of any one of embodiments A1-A8, whereinthe modification comprises a 2′-O-methyl (2′-O-Me) modified nucleotide.

Embodiment A10 is the guide RNA of any one of embodiments A1-A9, whereinthe modification comprises a phosphorothioate (PS) bond betweennucleotides.

Embodiment A11 is the guide RNA of any one of embodiments A1-A10,wherein the modification comprises a 2′-fluor (2′F) modified nucleotide.

Embodiment A12 is the guide RNA of any one of embodiments A1 or A3-A11,further comprising the nucleotide sequence of SEQ ID NO: 171.

Embodiment A13 is the guide RNA of embodiment A12, modified according tothe pattern of nucleotide sequence of SEQ ID NO: 405.

Embodiment A14 is the guide RNA of any one of embodiments A1 or A3-A11,further comprising the nucleotide sequence of SEQ ID NO: 173.

Embodiment A15 is the guide RNA of embodiment A14, modified according tothe pattern of SEQ ID NO: 248-255 or 450.

Embodiment A16 is the guide RNA of any one of embodiments A12-A15,wherein the guide sequence is SEQ ID NO: 15.

Embodiment A17 is the guide RNA of any one of embodiments A12-A15,wherein the guide sequence is SEQ ID NO: 8.

Embodiment A18 is the guide RNA of any one of embodiments A12-A15,wherein the guide sequence is SEQ ID NO: 41.

Embodiment A19 is the guide RNA of any one of embodiments A1 or A4-A11,wherein the guide RNA is modified according to the pattern of SEQ ID NO:300, wherein the N's are collectively the guide sequence of embodimentA1.

Embodiment A20 is the guide RNA of embodiment A16, wherein each N in SEQID NO: 300 is any natural or non-natural nucleotide.

Embodiment A21 is the guide RNA of embodiment A19, wherein the guidesequence is SEQ ID NO: 15 and the guide RNA is modified according tomG*mG*mA* UUGCGUAUGGGACACAAGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU, wherein “mA,” “mC,” “mU,” or“mG” denote a nucleotide that has been modified with 2′-O-Me, a *denotes a phosphorothioate bond, and an N is a natural nucleotide.

Embodiment A22 is the guide RNA of embodiment A19, wherein the guidesequence is SEQ ID NO: 8 and the guide RNA is modified according tomU*mA*mC*CCGGGAGUUGACUUUGGGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU, wherein “mA,” “mC,”“mU,” or “mG” denote a nucleotide that has been modified with 2′-O-Me,a * denotes a phosphorothioate bond, and N is a natural nucleotide.

Embodiment A23 is the guide RNA of embodiment A19, wherein the guidesequence is SEQ ID NO: 41 and the guide RNA is modified according tomU*mA*mU*UAUCAAAUCACAUUACCGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU, wherein “mA,” “mC,”“mU,” or “mG” denote a nucleotide that has been modified with 2′-O-Me,a * denotes a phosphorothioate bond, and N is a natural nucleotide.

Embodiment A24 is a composition comprising a guide RNA of any one ofembodiments A1-A23.

Embodiment A25 is a composition of embodiment A24, further comprising anRNA-guided DNA binding agent or nucleic acid encoding an RNA-guided DNAbinding agent.

Embodiment A26 is the composition of embodiment A25, wherein the nucleicacid encoding an RNA-guided DNA binding agent comprises an mRNAcomprising an open reading frame (ORF) encoding an RNA guided DNAbinding agent.

Embodiment A27 is the composition of embodiment A25 or A26, wherein theRNA-guided DNA binding agent is Cas9.

Embodiment A28 is the composition of embodiment A27, wherein the Cas9 isS. pyogenes Cas9.

Embodiment A29 is the composition of any one of embodiments A26-A28,wherein the ORF is a modified ORF.

Embodiment A30 is the composition of any one of embodiments A24-A29,further comprising a pharmaceutical excipient.

Embodiment A31 is the composition of any one of embodiments A24-A30,wherein the guide RNA is associated with a lipid nanoparticle (LNP).

Embodiment A32 is the composition of embodiment A31, wherein the LNPcomprises a cationic lipid.

Embodiment A33 is the composition of embodiment A32, wherein thecationic lipid is(9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate, also called3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate.

Embodiment A34 is the composition of any one of embodiments A31-A33,wherein the LNP comprises(9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate, also called3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate, DSPC, cholesterol, and PEG2k-DMG.

Embodiment A35 is a pharmaceutical composition comprising a guide RNA ofany one of embodiments A1-A23 or composition of any one of embodimentsA24-A34.

Embodiment A36 is a pharmaceutical composition comprising or use of aguide RNA of any one of embodiments A1-A23 or composition of any one ofembodiments A24-A34 for inducing a double stranded break or a singlestranded break within a KLKB1 gene in a cell or reducing expression ofKLKB1 in a cell.

Embodiment A37 is the pharmaceutical composition or use of embodimentA36, for reducing expression of the KLKB1 gene in a cell or subject.

Embodiment A38 is a pharmaceutical composition comprising or use of aguide RNA of any one of embodiments A1-A23 or composition of any one ofembodiments A24-A34 for treating a subject having hereditary angioedema(HAE).

Embodiment A39 is the pharmaceutical composition or use of embodimentA38, comprising reducing the frequency and/or severity of HAE attacks.

Embodiment A40 is a pharmaceutical composition comprising or use of aguide RNA of any one of embodiments A1-A23 or composition of any one ofembodiments A24-A34 for treating or preventing angioedema associatedwith HAE, bradykinin production and accumulation, bradykinin-inducedswelling, angioedema obstruction of the airway, or asphyxiation.

Embodiment A41 is a pharmaceutical composition or use of a guide RNA ofany one of embodiments A1-A23 or composition of any one of embodimentsA24-A34 for reducing total plasma kallikrein activity or reducingprekallikrein and/or kallikrein levels in a subject.

Embodiment A42 is the pharmaceutical composition or use of embodimentA41, wherein the total plasma kallikrein activity is reduced by morethan 60%.

Embodiment A43 is a method or inducing a double stranded break or asingle stranded break within a KLKB1 gene in a cell or reducingexpression of KLKB1 in a cell comprising contacting a cell with a guideRNA of any one of embodiments A1-A23 or composition of any one ofembodiments A24-A34.

Embodiment A44 is the method of embodiment A43, wherein the cell is in asubject.

Embodiment A45 is a method of treating a subject having hereditaryangioedema (HAE) comprising administering a guide RNA of any one ofembodiments A1-A23 or composition of any one of embodiments A24-34thereby treating the subject.

Embodiment A46 is the method of embodiment A45, wherein treating thesubject comprises reducing the frequency and/or severity of HAE attacks.

Embodiment A47 is a method of treating or preventing angioedemaassociated with HAE, bradykinin production and accumulation,bradykinin-induced swelling, angioedema obstruction of the airway, orasphyxiation comprising administering to the subject a guide RNA of anyone of embodiments A1-A23 or composition of any one of embodimentsA24-A34, thereby treating or preventing angioedema associated with HAE,bradykinin production and accumulation, bradykinin-induced swelling,angioedema obstruction of the airway, or asphyxiation in the subject.

Embodiment A48 is a method of reducing total plasma kallikrein activityin a subject comprising administrating a guide RNA of any one ofembodiments A1-A23 or composition of any one of embodiments A24-A34,thereby reducing total plasma kallikrein activity in a subject.

Embodiment A49 is the method of embodiment A48, wherein the total plasmakallikrein activity is reduced by more than 60% in the subject.

Embodiment A50 is the use of a guide RNA of any one of embodimentsA1-A23 or composition of any one of embodiments A24-A34 in thepreparation of a medicament for practicing any of the methods ofembodiments A43-A49.

Additional embodiments are provided herein.

Embodiment 1 is a method of inducing a double-stranded break (DSB) or asingle-stranded break (SSB) within the KLKB1 gene, comprising deliveringa composition to a cell, wherein the composition comprises:

-   -   a. a guide RNA comprising        -   i. a guide sequence selected from SEQ ID NOs: 1-149; or        -   ii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence selected from SEQ ID NOs: 1-149; or        -   iii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from SEQ ID NOs: 1-149; or        -   iv. a guide sequence comprising any one of SEQ ID NOs: 1, 7,            8, 15, 26, 27, 28, 41, 42, 46, 51, 52, 53, 56, 69, 71; or        -   v. a guide sequence comprising any one of SEQ ID Nos: 8, 15,            41, 51, 69; or        -   vi. a sequence that comprises 15 consecutive nucleotides f10            nucleotides of a genomic coordinate listed in Table 1; or        -   vii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence from (vi); or        -   viii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from (vi); and optionally    -   b. an RNA-guided DNA binding agent or a nucleic acid encoding an        RNA-guided DNA binding agent.

Embodiment 2 is a method of reducing the expression of the KLKB1 genecomprising delivering a composition to a cell, wherein the compositioncomprises:

-   -   a. a guide RNA comprising        -   i. a guide sequence selected from SEQ ID NOs: 1-149; or        -   ii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence selected from SEQ ID NOs: 1-149; or        -   iii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from SEQ ID NOs: 1-149; or        -   iv. a guide sequence comprising any one of SEQ ID NOs: 1, 7,            8, 15, 26, 27, 28, 41, 42, 46, 51, 52, 53, 56, 69, 71; or        -   v. a guide sequence comprising any one of SEQ ID Nos: 8, 15,            41, 51, 69; or        -   vi. a sequence that comprises 15 consecutive nucleotides f10            nucleotides of a genomic coordinate listed in Table 1; or        -   vii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence from (vi); or        -   viii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from (vi); and optionally    -   b. an RNA-guided DNA binding agent or a nucleic acid encoding an        RNA-guided DNA binding agent.

Embodiment 3 is a method of treating or preventing hereditary angioedema(HAE) comprising administering a composition to a subject in needthereof, wherein the composition comprises:

-   -   a. a guide RNA comprising        -   i. a guide sequence selected from SEQ ID NOs: 1-149; or        -   ii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence selected from SEQ ID NOs: 1-149; or        -   iii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from SEQ ID NOs: 1-149; or        -   iv. a guide sequence comprising any one of SEQ ID NOs: 1, 7,            8, 15, 26, 27, 28, 41, 42, 46, 51, 52, 53, 56, 69, 71; or        -   v. a guide sequence comprising any one of SEQ ID Nos: 8, 15,            41, 51, 69; or        -   vi. a sequence that comprises 15 consecutive nucleotides f10            nucleotides of a genomic coordinate listed in Table 1; or        -   vii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence from (vi); or        -   viii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from (vi); and optionally    -   b. an RNA-guided DNA binding agent or nucleic acid encoding an        RNA-guided DNA binding agent, thereby treating or preventing        HAE.

Embodiment 4 is a method of treating or preventing angioedema caused byor associated with HAE comprising administering a composition to asubject in need thereof, wherein the composition comprises:

-   -   a. a guide RNA comprising        -   i. a guide sequence selected from SEQ ID NOs: 1-149; or        -   ii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence selected from SEQ ID NOs: 1-149; or        -   iii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from SEQ ID NOs: 1-149; or        -   iv. a guide sequence comprising any one of SEQ ID NOs: 1, 7,            8, 15, 26, 27, 28, 41, 42, 46, 51, 52, 53, 56, 69, 71; or        -   v. a guide sequence comprising any one of SEQ ID Nos: 8, 15,            41, 51, 69; or        -   vi. a sequence that comprises 15 consecutive nucleotides f10            nucleotides of a genomic coordinate listed in Table 1; or        -   vii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence from (vi); or        -   viii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from (vi); and optionally    -   b. an RNA-guided DNA binding agent or nucleic acid encoding an        RNA-guided DNA binding agent, thereby treating or preventing        angioedema caused by or associated with HAE.

Embodiment 5 is a method of treating or preventing any one of bradykininproduction and accumulation, bradykinin-induced swelling, angioedemaobstruction of the airway, or asphyxiation comprising administering acomposition to a subject in need thereof, wherein the compositioncomprises:

-   -   a. a guide RNA comprising        -   i. a guide sequence selected from SEQ ID NOs: 1-149; or        -   ii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence selected from SEQ ID NOs: 1-149; or        -   iii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from SEQ ID NOs: 1-149; or        -   iv. a guide sequence comprising any one of SEQ ID NOs: 1, 7,            8, 15, 26, 27, 28, 41, 42, 46, 51, 52, 53, 56, 69, 71; or        -   v. a guide sequence comprising any one of SEQ ID Nos: 8, 15,            41, 51, 69; or        -   vi. a sequence that comprises 15 consecutive nucleotides f10            nucleotides of a genomic coordinate listed in Table 1; or        -   vii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence from (vi); or        -   viii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from (vi); and optionally    -   b. an RNA-guided DNA binding agent or nucleic acid encoding an        RNA-guided DNA binding agent, thereby treating or preventing any        one of bradykinin production and accumulation,        bradykinin-induced swelling, angioedema obstruction of the        airway, or asphyxiation.

Embodiment 6 is a method of reducing the frequency and/or severity ofHAE attacks, comprising administering a composition to a subject in needthereof, wherein the composition comprises:

-   -   a. a guide RNA comprising        -   i. a guide sequence selected from SEQ ID NOs: 1-149; or        -   ii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence selected from SEQ ID NOs: 1-149; or        -   iii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from SEQ ID NOs: 1-149; or        -   iv. a guide sequence comprising any one of SEQ ID NOs: 1, 7,            8, 15, 26, 27, 28, 41, 42, 46, 51, 52, 53, 56, 69, 71; or        -   v. a guide sequence comprising any one of SEQ ID Nos: 8, 15,            41, 51, 69; or        -   vi. a sequence that comprises 15 consecutive nucleotides f10            nucleotides of a genomic coordinate listed in Table 1; or        -   vii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence from (vi); or        -   viii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from (vi); and optionally    -   b. an RNA-guided DNA binding agent or nucleic acid encoding an        RNA-guided DNA binding agent, thereby reducing the frequency        and/or severity of HAE attacks.

Embodiment 7 is a method for reducing the frequency and/or severity ofangioedema attacks, or achieving remission of angioedema attacks in asubject, comprising administering a composition to a subject in needthereof, wherein the composition comprises:

-   -   a. a guide RNA comprising        -   i. a guide sequence selected from SEQ ID NOs: 1-149; or        -   ii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence selected from SEQ ID NOs: 1-149; or        -   iii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from SEQ ID NOs: 1-149; or        -   iv. a guide sequence comprising any one of SEQ ID NOs: 1, 7,            8, 15, 26, 27, 28, 41, 42, 46, 51, 52, 53, 56, 69, 71; or        -   v. a guide sequence comprising any one of SEQ ID Nos: 8, 15,            41, 51, 69; or        -   vi. a sequence that comprises 15 consecutive nucleotides f10            nucleotides of a genomic coordinate listed in Table 1; or        -   vii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence from (vi); or        -   viii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from (vi); and optionally    -   b. an RNA-guided DNA binding agent or nucleic acid encoding an        RNA-guided DNA binding agent, thereby reducing the frequency        and/or severity of angioedema attacks or achieving remission of        angioedema attacks in a subject.

Embodiment 8 is a method of reducing total plasma kallikrein activity,comprising administering a composition to a subject in need thereof,wherein the composition comprises:

-   -   a. a guide RNA comprising        -   i. a guide sequence selected from SEQ ID NOs: 1-149; or        -   ii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence selected from SEQ ID NOs: 1-149; or        -   iii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from SEQ ID NOs: 1-149; or        -   iv. a guide sequence comprising any one of SEQ ID NOs: 1, 7,            8, 15, 26, 27, 28, 41, 42, 46, 51, 52, 53, 56, 69, 71; or        -   v. a guide sequence comprising any one of SEQ ID Nos: 8, 15,            41, 51, 69; or        -   vi. a sequence that comprises 15 consecutive nucleotides f10            nucleotides of a genomic coordinate listed in Table 1; or        -   vii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence from (vi); or        -   viii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from (vi); and optionally    -   b. an RNA-guided DNA binding agent or nucleic acid encoding an        RNA-guided DNA binding agent, thereby achieving remission of        angioedema attacks in a subject, wherein total plasma kallikrein        activity is reduced.

Embodiment 9 is the method of embodiment 8, further comprising anactivation step to convert prekallikrein to its active form, pKal.

Embodiment 10 is the method of embodiment 8, wherein the total plasmakallikrein activity is reduced by more than 60%, more than 85%, or morethan 60-80%.

Embodiment 11 is a method of reducing total plasma kallikrein levels,comprising administering a composition to a subject in need thereof,wherein the composition comprises:

-   -   a. a guide RNA comprising        -   i. a guide sequence selected from SEQ ID NOs: 1-149; or        -   ii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence selected from SEQ ID NOs: 1-149; or        -   iii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from SEQ ID NOs: 1-149; or        -   iv. a guide sequence comprising any one of SEQ ID NOs: 1, 7,            8, 15, 26, 27, 28, 41, 42, 46, 51, 52, 53, 56, 69, 71; or        -   v. a guide sequence comprising any one of SEQ ID Nos: 8, 15,            41, 51, 69; or        -   vi. a sequence that comprises 15 consecutive nucleotides f10            nucleotides of a genomic coordinate listed in Table 1; or        -   vii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence from (vi); or        -   viii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from (vi); and optionally    -   b. an RNA-guided DNA binding agent or nucleic acid encoding an        RNA-guided DNA binding agent, thereby total plasma kallikrein        levels.

Embodiment 12 is a method of reducing prekallikrein and/or kallikreinlevels, comprising administering a composition to a subject in needthereof, wherein the composition comprises:

-   -   a. a guide RNA comprising        -   i. a guide sequence selected from SEQ ID NOs: 1-149; or        -   ii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence selected from SEQ ID NOs: 1-149; or        -   iii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from SEQ ID NOs: 1-149; or        -   iv. a guide sequence comprising any one of SEQ ID NOs: 1, 7,            8, 15, 26, 27, 28, 41, 42, 46, 51, 52, 53, 56, 69, 71; or        -   v. a guide sequence comprising any one of SEQ ID Nos: 8, 15,            41, 51, 69; or        -   vi. a sequence that comprises 15 consecutive nucleotides f10            nucleotides of a genomic coordinate listed in Table 1; or        -   vii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence from (vi); or        -   viii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from (vi); and optionally    -   b. an RNA-guided DNA binding agent or nucleic acid encoding an        RNA-guided DNA binding agent, thereby reducing prekallikrein        and/or kallikrein.

Embodiment 13 is the method of any one of the preceding embodiments,wherein there is a dose dependent increase in percent editing.

Embodiment 14 is the method of embodiment 13, wherein there is a dosedependent reduction in total plasma kallikrein levels.

Embodiment 15 is the method of embodiment 13 or 14, wherein there is adose dependent reduction in plasma kallikrein activity.

Embodiment 16 is the method of any one of the preceding embodimentswherein the effect is durable for at least 1 month, 2 months, 4 months,6 months, 1 year, 2 years, 5 years, 10 years or more after theadministration.

Embodiment 17 is the method of any one of the preceding embodimentswherein the effect is durable for at least 6 months.

Embodiment 18 is the method of any one of the preceding embodimentswherein the effect is durable for at least 1 year.

Embodiment 19 is the method of embodiment 6, wherein the frequency ofHAE attacks is reduced.

Embodiment 20 is the method of embodiment 19, wherein the frequency isreduced by at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 60-80%, or at least 40-90%.

Embodiment 21 is the method of embodiment 20, wherein the frequency isreduced by at least 60-80%.

Embodiment 22 is the method of embodiment 20, wherein the frequency isreduced by at least 40-90%.

Embodiment 23 is the method of any one of the preceding embodiments,wherein the effect is durable for at least 1 month, 2 months, 4 months,6 months, 1 year, 2 years, 5 years, 10 years or more after theadministration.

Embodiment 24 is the method of any one of the preceding embodiments,wherein the effect is durable for at least 6 months after theadministration.

Embodiment 25 is the method of any one of the preceding embodiments,wherein the effect is durable for at least 1 year after theadministration.

Embodiment 26 is the method of any one of the preceding embodiments,wherein the effect is compared to a basal level.

Embodiment 27 is the method of any one of the preceding embodiments,wherein the effect is compared to a subject's basal level.

Embodiment 28 is the method of any one of the preceding embodiments,wherein an RNA-guided DNA binding agent or nucleic acid encoding anRNA-guided DNA binding agent is administered.

Embodiment 29 is a composition comprising:

-   -   a. a guide RNA comprising        -   i. a guide sequence selected from SEQ ID NOs: 1-149; or        -   ii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence selected from SEQ ID NOs: 1-149; or        -   iii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from SEQ ID NOs: 1-149; or        -   iv. a guide sequence comprising any one of SEQ ID NOs: 1, 7,            8, 15, 26, 27, 28, 41, 42, 46, 51, 52, 53, 56, 69, 71; or        -   v. a guide sequence comprising any one of SEQ ID Nos: 8, 15,            41, 51, 69; or        -   vi. a sequence that comprises 15 consecutive nucleotides f10            nucleotides of a genomic coordinate listed in Table 1; or        -   vii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence from (vi); or        -   viii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from (vi); and optionally    -   b. an RNA-guided DNA binding agent or nucleic acid encoding an        RNA-guided DNA binding agent.

Embodiment 30 is a composition comprising a short-single guide RNA(short-sgRNA), comprising:

-   -   a. a guide sequence comprising:        -   i. a guide sequence selected from SEQ ID NOs: 1-149; or        -   ii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence selected from SEQ ID NOs: 1-149; or        -   iii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from SEQ ID NOs: 1-149; or        -   iv. a guide sequence comprising any one of SEQ ID NOs: 1, 7,            8, 15, 26, 27, 28, 41, 42, 46, 51, 52, 53, 56, 69, 71; or        -   v. a guide sequence comprising any one of SEQ ID Nos: 8, 15,            41, 51, 69; or        -   vi. a sequence that comprises 15 consecutive nucleotides f10            nucleotides of a genomic coordinate listed in Table 1; or        -   vii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence from (vi); or        -   viii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from (vi); and    -   b. a conserved portion of an sgRNA comprising a hairpin region,        wherein the hairpin region lacks at least 5-10 nucleotides and        optionally wherein the short-sgRNA comprises one or more of a 5′        end modification and a 3′ end modification.

Embodiment 31. The composition of embodiment 29, comprising the sequenceof SEQ ID NO: 202.

Embodiment 32 is the composition of embodiment 29 or embodiment 30,comprising a 5′ end modification.

Embodiment 33 is the composition of any one of embodiments 29-32,wherein the short-sgRNA comprises a 3′ end modification.

Embodiment 34 is the composition of any one of embodiments 29-33,wherein the short-sgRNA comprises a 5′ end modification and a 3′ endmodification.

Embodiment 35 is the composition of any one of embodiments 29-34,wherein the short-sgRNA further comprises a 3′ tail.

Embodiment 36 is the composition of embodiment 35, wherein the 3′ tailcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

Embodiment 37 is the composition of embodiment 35, wherein the 3′ tailcomprises about 1-2, 1-3, 1-4, 1-5, 1-7, 1-10, at least 1-2, at least1-3, at least 1-4, at least 1-5, at least 1-7, or at least 1-10nucleotides.

Embodiment 38 is the composition of any one of embodiments 29-37,wherein the short-sgRNA does not comprise a 3′ tail.

Embodiment 39 is the composition of any one of embodiments 29-38,comprising a modification in the hairpin region.

Embodiment 40 is the composition of any one of embodiments 29-39,comprising a 3′ end modification, and a modification in the hairpinregion.

Embodiment 41 is the composition of any one of embodiments 29-40,comprising a 3′ end modification, a modification in the hairpin region,and a 5′ end modification.

Embodiment 42 is the composition of any one of embodiments 29-41,comprising a 5′ end modification, and a modification in the hairpinregion.

Embodiment 43 is the composition of any one of embodiments 29-42,wherein the hairpin region lacks at least 5 consecutive nucleotides.

Embodiment 44 is the composition of any one of embodiments 29-43,wherein the at least 5-10 lacking nucleotides:

-   -   a. are within hairpin 1;    -   b. are within hairpin 1 and the “N” between hairpin 1 and        hairpin 2;    -   c. are within hairpin 1 and the two nucleotides immediately 3′        of hairpin 1;    -   d. include at least a portion of hairpin 1;    -   e. are within hairpin 2;    -   f. include at least a portion of hairpin 2;    -   g. are within hairpin 1 and hairpin 2;    -   h. include at least a portion of hairpin 1 and include the “N”        between hairpin 1 and hairpin 2;    -   i. include at least a portion of hairpin 2 and include the “N”        between hairpin 1 and hairpin 2;    -   j. include at least a portion of hairpin 1, include the “N”        between hairpin 1 and hairpin 2, and include at least a portion        of hairpin 2;    -   k. are within hairpin 1 or hairpin 2, optionally including the        “N” between hairpin 1 and hairpin 2;    -   l. are consecutive;    -   m. are consecutive and include the “N” between hairpin 1 and        hairpin 2;    -   n. are consecutive and span at least a portion of hairpin 1 and        a portion of hairpin 2;    -   o. are consecutive and span at least a portion of hairpin 1 and        the “N” between hairpin 1 and hairpin 2;    -   p. are consecutive and span at least a portion of hairpin 1 and        two nucleotides immediately 3′ of hairpin 1;    -   q. consist of 5-10 nucleotides;    -   r. consist of 6-10 nucleotides;    -   s. consist of 5-10 consecutive nucleotides;    -   t. consist of 6-10 consecutive nucleotides; or    -   u. consist of nucleotides 54-58 of SEQ ID NO: 400.

Embodiment 45 is the composition of any one of embodiments 29-44,comprising a conserved portion of an sgRNA comprising a nexus region,wherein the nexus region lacks at least one nucleotide.

Embodiment 46 is the composition of embodiment 45, wherein thenucleotides lacking in the nexus region comprise any one or more of:

-   -   a. at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in the        nexus region;    -   b. at least or exactly 1-2 nucleotides, 1-3 nucleotides, 1-4        nucleotides, 1-5 nucleotides, 1-6 nucleotides, 1-10 nucleotides,        or 1-15 nucleotides in the nexus region; and    -   c. each nucleotide in the nexus region.

Embodiment 47 is a composition comprising a modified single guide RNA(sgRNA) comprising

-   -   a. a guide sequence comprising:        -   i. a guide sequence selected from SEQ ID NOs: 1-149; or        -   ii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence selected from SEQ ID NOs: 1-149; or        -   iii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from SEQ ID NOs: 1-149; or        -   iv. a guide sequence comprising any one of SEQ ID NOs: 1, 7,            8, 15, 26, 27, 28, 41, 42, 46, 51, 52, 53, 56, 69, 71; or        -   v. a guide sequence comprising any one of SEQ ID Nos: 8, 15,            41, 51, 69; or        -   vi. a sequence that comprises 15 consecutive nucleotides f10            nucleotides of a genomic coordinate listed in Table 1; or        -   vii. at least 17, 18, 19, or 20 contiguous nucleotides of a            sequence from (vi); or        -   viii. a guide sequence that is at least 95%, 90%, or 85%            identical to a sequence selected from (vi); and

further comprising

-   -   b. one or more modifications selected from:        -   1. a YA modification at one or more guide region YA sites;        -   2. a YA modification at one or more conserved region YA            sites;        -   3. a YA modification at one or more guide region YA sites            and at one or more conserved region YA sites;        -   4. i) a YA modification at two or more guide region YA            sites;            -   ii) a YA modification at one or more of conserved region                YA sites 2, 3, 4, and 10; and            -   iii) a YA modification at one or more of conserved                region YA sites 1 and 8; or        -   5. i) a YA modification at one or more guide region YA            sites, wherein the guide region YA site is at or after            nucleotide 8 from the 5′ end of the 5′ terminus;            -   ii) a YA modification at one or more of conserved region                YA sites 2, 3, 4, and 10; and optionally;            -   iii) a YA modification at one or more of conserved                region YA sites 1 and 8; or        -   6. i) a YA modification at one or more guide region YA            sites, wherein the guide region YA site is within 13            nucleotides of the 3′ terminal nucleotide of the guide            region;            -   ii) a YA modification at one or more of conserved region                YA sites 2, 3, 4, and 10; and            -   iii) a YA modification at one or more of conserved                region YA sites 1 and 8; or        -   7. i) a 5′ end modification and a 3′ end modification;            -   ii) a YA modification at one or more of conserved region                YA sites 2, 3, 4, and 10; and            -   iii) a YA modification at one or more of conserved                region YA sites 1 and 8; or        -   8. i) a YA modification at a guide region YA site, wherein            the modification of the guide region YA site comprises a            modification that at least one nucleotide located 5′ of the            guide region YA site does not comprise;            -   ii) a YA modification at one or more of conserved region                YA sites 2, 3, 4, and 10; and            -   iii) a YA modification at one or more of conserved                region YA sites 1 and 8; or        -   9. i) a YA modification at one or more of conserved region            YA sites 2, 3, 4, and 10; and            -   ii) a YA modification at conserved region YA sites 1 and                8; or        -   10. i) a YA modification at one or more guide region YA            sites, wherein the YA site is at or after nucleotide 8 from            the 5′ terminus;            -   ii) a YA modification at one or more of conserved region                YA sites 2, 3, 4, and 10; and            -   iii) a modification at one or more of H1-1 and H2-1; or        -   11. i) a YA modification at one or more of conserved region            YA sites 2, 3, 4, and 10; ii) a YA modification at one or            more of conserved region YA sites 1, 5, 6, 7, 8, and 9;            and iii) a modification at one or more of H1-1 and H2-1; or        -   12. i) a modification, such as a YA modification, at one or            more nucleotides located at or after nucleotide 6 from the            5′ terminus;            -   ii) a YA modification at one or more guide sequence YA                sites;            -   iii) a modification at one or more of B3, B4, and B5,                wherein B6 does not comprise a 2′-OMe modification or                comprises a modification other than 2′-OMe;            -   iv) a modification at LS10, wherein LS10 comprises a                modification other than 2′-fluoro; and/or            -   v) a modification at N2, N3, N4, N5, N6, N7, N10, or                N11; and wherein at least one of the following is true:                -   i. a YA modification at one or more guide region YA                    sites;                -   ii. a YA modification at one or more conserved                    region YA sites;                -   iii. a YA modification at one or more guide region                    YA sites and at one or more conserved region YA                    sites;                -   iv. at least one of nucleotides 8-11, 13, 14, 17, or                    18 from the 5′ end of the 5′ terminus does not                    comprise a 2′-fluoro modification;                -   v. at least one of nucleotides 6-10 from the 5′ end                    of the 5′ terminus does not comprise a                    phosphorothioate linkage;                -   vi. at least one of B2, B3, B4, or B5 does not                    comprise a 2′-OMe modification;                -   vii. at least one of LS1, LS8, or LS10 does not                    comprise a 2′-OMe modification;                -   viii. at least one of N2, N3, N4, N5, N6, N7, N10,                    N11, N16, or N17 does not comprise a 2′-OMe                    modification;                -   ix. H1-1 comprises a modification;                -   x. H2-1 comprises a modification; or                -   xi. at least one of H1-2, H1-3, H1-4, H1-5, H1-6,                    H1-7, H1-8, H1-9, H1-10, H2-1, H2-2, H2-3, H2-4,                    H2-5, H2-6, H2-7, H2-8, H2-9, H2-10, H2-11, H2-12,                    H2-13, H2-14, or H2-15 does not comprise a                    phosphorothioate linkage.

Embodiment 48 is the composition of embodiment 47, comprising SEQ ID NO:450.

Embodiment 49 is the composition of any one of embodiments 29-48, foruse in inducing a double-stranded break (DSB) or a single-stranded breakwithin the KLKB1 gene in a cell or subject.

Embodiment 50 is the composition of any one of embodiments 29-48, foruse in reducing the expression of the KLKB1 gene in a cell or subject.

Embodiment 51 is the composition of any one of embodiments 29-48, foruse in treating or preventing HAE in a subject.

Embodiment 52 is the composition of any one of embodiments 29-48, foruse in reducing serum and/or plasma bradykinin concentration in asubject.

Embodiment 53 is the composition of any one of embodiments 29-48, foruse in reducing bradykinin-mediated vasodilation concentration in asubject.

Embodiment 54 is the composition of any one of embodiments 29-48, foruse in treating or preventing bradykinin production and accumulation,bradykinin-mediated vasodilation, swelling, or angioedema, obstructionof the airway, or asphyxiation.

Embodiment 55 is the composition of any one of embodiments 29-48, foruse in treating or preventing angioedema caused by or associated withHAE.

Embodiment 56 is the composition of any one of embodiments 29-48, foruse in reducing the frequency of angioedema attacks.

Embodiment 57 is the composition of any one of embodiments 29-48, foruse in reducing the severity of angioedema attacks.

Embodiment 58 is the composition of any one of embodiments 29-48, foruse in reducing the frequency and/or severity of attacks.

Embodiment 59 is the composition of any one of embodiments 29-48, foruse in achieving remission of angioedema attacks.

Embodiment 60 is the composition of any one of embodiments 29-48, foruse in reducing the frequency and/or severity of HAE attacks.

Embodiment 61 is the composition of embodiment 60, for use in reducingthe frequency of HAE attacks.

Embodiment 62 is the composition of embodiment 61, wherein the frequencyis reduced by at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 60-80%, or at least 40-90%.

Embodiment 63 is the method of embodiment 61, wherein the frequency isreduced by at least 60-80%.

Embodiment 64 is the method of embodiment 61, wherein the frequency isreduced by at least 40-90%.

Embodiment 65 is the composition of embodiment 60, for use in reducingtotal plasma kallikrein activity.

Embodiment 66 is the composition of embodiment 60, for use in reducingtotal plasma kallikrein levels.

Embodiment 67 is the composition of embodiment 60, for use in reducingprekallikrein and/or kallikrein levels.

Embodiment 68 is the composition of any one of embodiments 65-67,wherein there is a dose dependent increase in percent editing.

Embodiment 69 is the composition of any one of embodiments 65-68,wherein there is a dose dependent reduction in total plasma kallikreinlevels.

Embodiment 70 is the composition of any one of embodiments 65-69,wherein there is a dose dependent reduction in plasma kallikreinactivity.

Embodiment 71 is the composition of any one of embodiments 29-70,wherein the effect is durable for at least 1 month, 2 months, 4 months,6 months, 1 year, 2 years, 5 years, 10 years or more after theadministration.

Embodiment 72 is the composition of any one of embodiments 29-71,wherein the effect is durable for at least 6 months.

Embodiment 73 is the composition of any one of embodiments 29-72,wherein the effect is durable for at least 1 year.

Embodiment 74 is the method of any of embodiments 1-28, furthercomprising:

-   -   a. inducing a double-stranded break (DSB) within the KLKB1 gene        in a cell or subject;    -   b. reducing the expression of the KLKB1 gene in a cell or        subject;    -   c. treating or preventing HAE in a subject;    -   d. reducing serum and/or plasma bradykinin concentration in a        subject;    -   e. reducing bradykinin production;    -   f. reducing bradykinin-mediated vasodilation;    -   g. treating or preventing bradykinin-mediated swelling and        angioedema; and/or    -   h. treating or preventing obstruction of the airway or        asphyxiation caused by swelling.

Embodiment 75 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the composition decreases KLKB1mRNA production.

Embodiment 76 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the composition decreasesprekallikrein protein levels in plasma or serum.

Embodiment 77 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the composition decreases totalkallikrein (prekallikrein and pKal) protein levels in plasma or serum.

Embodiment 78 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the composition decreases theproportion of circulating cleaved HMWK (cHMWK) compared to total HMWK incitrated serum or citrated plasma.

Embodiment 79 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the composition reduces asubject's proportion of cHMWK in citrated plasma to below 30% of totalHMWK.

Embodiment 80 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the composition decreases thespontaneous pKal activity in serum or plasma.

Embodiment 81 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the composition decreaseskallikrein activity.

Embodiment 82 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the kallikrein activity comprisestotal kallikrein activity, prekallikrein activity, and/or pKal activity.

Embodiment 83 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the composition reduces asubject's pKal activity by at least about 40% prior to the method or useof the composition.

Embodiment 84 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the composition reduces asubject's pKal activity by at least about 50% prior to the method or useof the composition.

Embodiment 85 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the composition reduces asubject's pKal activity by at least about 60% prior to the method or useof the composition.

Embodiment 86 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the composition reduces asubject's pKal activity to less than about 40% of basal levels.

Embodiment 87 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the composition reduces asubject's pKal activity to about 40-50% of basal levels.

Embodiment 88 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the composition reduces asubject's pKal activity to 20-40% or 20-50% of basal levels.

Embodiment 89 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the composition increases serumand/or plasma bradykinin levels.

Embodiment 90 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the composition results inediting of the KLKB1 gene.

Embodiment 91 is the method, composition, or composition for use ofembodiment 90, wherein the editing is calculated as a percentage of thepopulation that is edited (percent editing).

Embodiment 92 is the method, composition, or composition for use ofembodiment 91, wherein the percent editing is between 30 and 99% of thepopulation.

Embodiment 93 is the method, composition, or composition for use ofembodiment 91, wherein the percent editing is between 30 and 35%, 35 and40%, 40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60 and 65%, 65 and70%, 70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90 and 95%, or 95and 99% of the population.

Embodiment 94 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the composition reduces serumand/or plasma bradykinin concentration.

Embodiment 95 is the method, composition, or composition for use of anyone the preceding embodiments, wherein the composition reduces serumand/or plasma bradykinin concentration, and wherein a reduction in serumand/or plasma bradykinin results in decreased swelling in organ tissues,including limbs, face, GI tract, or airway.

Embodiment 96 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the guide sequence is selectedfrom

-   -   a. SEQ ID NOs: 1-149; or    -   b. SEQ ID NOs: 1, 7, 8, 15, 26, 27, 28, 41, 42, 46, 51, 52, 53,        56, 69, 71; or    -   c. any one of SEQ ID Nos: 8, 15, 41, 51, 69; or    -   d. a sequence that comprises 15 consecutive nucleotides ±10        nucleotides of a genomic coordinate listed in Table 1; or    -   e. at least 17, 18, 19, or 20 contiguous nucleotides of a        sequence from (d); or    -   f. a guide sequence that is at least 95%, 90%, or 85% identical        to a sequence selected from (d).

Embodiment 97 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprises asgRNA comprising:

-   -   a. SEQ ID NOs: 1, 7, 8, 15, 26, 27, 28, 41, 42, 46, 51, 52, 53,        56, 69, 71; or    -   b. any one of SEQ ID Nos: 8, 15, 41, 51, 69; or    -   c. a sequence that comprises 15 consecutive nucleotides f10        nucleotides of a genomic coordinate listed in Table 1; or    -   d. at least 17, 18, 19, or 20 contiguous nucleotides of a        sequence from (c); or    -   e. a guide sequence that is at least 95%, 90%, or 85% identical        to a sequence selected from (c).

Embodiment 98 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the target sequence is in exon1, exon 3, exon 4, exon 5, exon 6, or exon 8, exon 9, exon 10, exon 11,exon 12, exon 13, exon 14, or exon 15 of the human KLKB1 gene.

Embodiment 99 is the method, composition for use, or composition ofembodiment 98, wherein the target sequence is in exon 1 of the humanKLKB1 gene.

Embodiment 100 is the method, composition for use, or composition ofembodiment 98, wherein the target sequence is in exon 3 of the humanKLKB1 gene.

Embodiment 101 is the method, composition for use, or composition ofembodiment 98, wherein the target sequence is in exon 4 of the humanKLKB1 gene.

Embodiment 102 is the method, composition for use, or composition ofembodiment 98, wherein the target sequence is in exon 5 of the humanKLKB1 gene.

Embodiment 103 is the method, composition for use, or composition ofembodiment 98, wherein the target sequence is in exon 6 of the humanKLKB1 gene.

Embodiment 104 is the method, composition for use, or composition ofembodiment 98, wherein the target sequence is in exon 8 of the humanKLKB1 gene.

Embodiment 105 is the method, composition for use, or composition ofembodiment 98, wherein the target sequence is in exon 9 of the humanKLKB1 gene.

Embodiment 106 is the method, composition for use, or composition ofembodiment 98, wherein the target sequence is in exon 10 of the humanKLKB1 gene.

Embodiment 107 is the method, composition for use, or composition ofembodiment 98, wherein the target sequence is in exon 11 of the humanKLKB1 gene.

Embodiment 108 is the method, composition for use, or composition ofembodiment 98, wherein the target sequence is in exon 12 of the humanKLKB1 gene.

Embodiment 109 is the method, composition for use, or composition ofembodiment 98, wherein the target sequence is in exon 13 of the humanKLKB1 gene.

Embodiment 110 is the method, composition for use, or composition ofembodiment 98, wherein the target sequence is in exon 14 of the humanKLKB1 gene.

Embodiment 111 is the method, composition for use, or composition ofembodiment 98, wherein the target sequence is in exon 15 of the humanKLKB1 gene.

Embodiment 112 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the guide sequence iscomplementary to a target sequence in the positive strand of KLKB1.

Embodiment 113 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the guide sequence iscomplementary to a target sequence in the negative strand of KLKB1.

Embodiment 114 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the first guide sequence iscomplementary to a first target sequence in the positive strand of theKLKB1 gene, and wherein the composition further comprises a second guidesequence that is complementary to a second target sequence in thenegative strand of the KLKB1 gene.

Embodiment 115 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the guide RNA comprises aguide sequence selected from any one of SEQ ID Nos 1-149 and furthercomprises a nucleotide sequence of SEQ ID NO: 170, wherein thenucleotides of SEQ ID NO: 170 follow the guide sequence at its 3′ end.

Embodiment 116 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the guide RNA comprises aguide sequence selected from any one of SEQ ID Nos: 1-149 and furthercomprises a nucleotide sequence of SEQ ID NO: 171, SEQ ID NO: 172, SEQID NO: 173, or any one of SEQ ID Nos: 400-450, wherein the nucleotidesof SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, or any one ofconserved portions of sgRNA from Table 4 follow the guide sequence atits 3′ end.

Embodiment 117 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the guide RNA is a singleguide RNA (sgRNA).

Embodiment 118 is the method, composition for use, or composition ofembodiment 117, wherein the sgRNA comprises a guide sequence comprisingany one of SEQ ID Nos: 8, 15, 41, 51, 69.

Embodiment 119 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the guide RNA is modifiedaccording to the pattern of SEQ ID NO: 300, wherein the N's arecollectively any one of the guide sequences of Table 1 (SEQ ID Nos:1-149).

Embodiment 120 is the method, composition for use, or composition ofembodiment 119, wherein each N in SEQ ID NO: 300 is any natural ornon-natural nucleotide, wherein the N's form the guide sequence, and theguide sequence targets Cas9 to the KLKB1 gene.

Embodiment 121 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the sgRNA comprises any one ofthe guide sequences of SEQ ID NOs: 1-149 and the nucleotides of SEQ IDNO: 171, SEQ ID NO: 172, SEQ ID NO: 173, or any of the conservedportions of sgRNA from Table 4, wherein the nucleotides of SEQ ID NO:171, SEQ ID NO: 172, SEQ ID NO: 173, or any of the conserved portions ofsgRNA from Table 4 follow the guide sequence at its 3′ end.

Embodiment 122 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the sgRNA comprises a guidesequence that is at least 95%, 90%, or 85% identical to a sequenceselected from SEQ ID Nos: 1-149.

Embodiment 123 is the method, composition for use, or composition ofembodiment 122, wherein the sgRNA comprises a sequence selected from SEQID Nos: 8, 15, 41, 51, 69.

Embodiment 124 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the guide RNA comprises atleast one modification.

Embodiment 125 is the method, composition for use, or composition ofembodiment 124, wherein the at least one modification includes a2′-O-methyl (2′-O-Me) modified nucleotide.

Embodiment 126 is the method, composition for use, or composition of anyone of embodiments 124-125, comprising a phosphorothioate (PS) bondbetween nucleotides.

Embodiment 127 is the method, composition for use, or composition of anyone of embodiments 124-126, comprising a 2′-fluoro (2′-F) modifiednucleotide.

Embodiment 128 is the method, composition for use, or composition of anyone of embodiments 124-127, comprising a modification at one or more ofthe first five nucleotides at the 5′ end of the guide RNA.

Embodiment 129 is the method, composition for use, or composition of anyone of embodiments 124-128, comprising a modification at one or more ofthe last five nucleotides at the 3′ end of the guide RNA.

Embodiment 130 is the method, composition for use, or composition of anyone of embodiments 124-129, comprising a PS bond between the first fournucleotides of the guide RNA.

Embodiment 131 is the method, composition for use, or composition of anyone of embodiments 124-130, comprising a PS bond between the last fournucleotides of the guide RNA.

Embodiment 132 is the method, composition for use, or composition of anyone of embodiments 124-131, comprising a 2′-O-Me modified nucleotide atthe first three nucleotides at the 5′ end of the guide RNA.

Embodiment 133 is the method, composition for use, or composition of anyone of embodiments 124-132, comprising a 2′-O-Me modified nucleotide atthe last three nucleotides at the 3′ end of the guide RNA.

Embodiment 134 is the method, composition for use, or composition of anyone of embodiments 124-133, wherein the guide RNA comprises the modifiednucleotides of SEQ ID NO: 300.

Embodiment 135 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition furthercomprises a pharmaceutically acceptable excipient.

Embodiment 136 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the guide RNA is associatedwith a lipid nanoparticle (LNP).

Embodiment 137 is the method, composition for use, or composition ofembodiment 136, wherein the LNP comprises a cationic lipid.

Embodiment 138 is the method, composition for use, or composition ofembodiment 137, wherein the cationic lipid is(9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate, also called3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate.

Embodiment 139 is the method, composition for use, or composition of anyone of embodiments 136-138, wherein the LNP comprises a neutral lipid.

Embodiment 140 is the method, composition for use, or composition ofembodiment 139, wherein the neutral lipid is DSPC.

Embodiment 141 is the method, composition for use, or composition of anyone of embodiments 136-140, wherein the LNP comprises a helper lipid.

Embodiment 142 is the method, composition for use, or composition ofembodiment 141, wherein the helper lipid is cholesterol.

Embodiment 143 is the method, composition for use, or composition of anyone of embodiments 136-142, wherein the LNP comprises a stealth lipid.

Embodiment 144 is the method, composition for use, or composition ofembodiment 143, wherein the stealth lipid is PEG2k-DMG.

Embodiment 145 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition furthercomprises an RNA-guided DNA binding agent.

Embodiment 146 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition furthercomprises an mRNA that encodes an RNA-guided DNA binding agent.

Embodiment 147 is the method, composition for use, or composition ofembodiment 145 or 146, wherein the RNA-guided DNA binding agent is Cas9.

Embodiment 148 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition is apharmaceutical formulation and further comprises a pharmaceuticallyacceptable carrier.

Embodiment 149 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprises asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 1.

Embodiment 150 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 2.

Embodiment 151 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 3.

Embodiment 152 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 4.

Embodiment 153 is the method, composition for use, or composition of anyone of embodiments 1-89, wherein the sequence selected from SEQ ID NOs:1-149 is SEQ ID NO: 5.

Embodiment 154 is the method, composition for use, or composition of anyone of embodiments 1-89, wherein the sequence selected from SEQ ID NOs:1-149 is SEQ ID NO: 6.

Embodiment 155 is the method, composition for use, or composition of anyone of embodiments 1-89, wherein the sequence selected from SEQ ID NOs:1-149 is SEQ ID NO: 7.

Embodiment 156 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprises asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 8.

Embodiment 157 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 9.

Embodiment 158 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 10.

Embodiment 159 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 11.

Embodiment 160 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 12.

Embodiment 161 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 13.

Embodiment 162 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 14.

Embodiment 163 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 15.

Embodiment 164 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 16.

Embodiment 165 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 17.

Embodiment 166 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 18.

Embodiment 167 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 19.

Embodiment 168 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 20.

Embodiment 169 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 21.

Embodiment 170 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 22.

Embodiment 171 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 23.

Embodiment 172 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 24.

Embodiment 173 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 25.

Embodiment 174 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 26.

Embodiment 175 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 27.

Embodiment 176 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 28.

Embodiment 177 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 29.

Embodiment 178 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 30.

Embodiment 179 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 31.

Embodiment 180 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 32.

Embodiment 181 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 33.

Embodiment 182 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 34.

Embodiment 183 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 35.

Embodiment 184 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 36.

Embodiment 185 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 37.

Embodiment 186 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 38.

Embodiment 187 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 39.

Embodiment 188 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 40.

Embodiment 189 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 41.

Embodiment 190 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 42.

Embodiment 191 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 43.

Embodiment 192 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 44.

Embodiment 193 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 45.

Embodiment 194 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 46.

Embodiment 195 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 47.

Embodiment 196 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 48.

Embodiment 197 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 49.

Embodiment 198 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 50.

Embodiment 199 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 51.

Embodiment 200 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 52.

Embodiment 201 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 53.

Embodiment 202 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 54.

Embodiment 203 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 55.

Embodiment 204 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 56.

Embodiment 205 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 57.

Embodiment 206 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 58.

Embodiment 207 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 59.

Embodiment 208 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 60.

Embodiment 209 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 61.

Embodiment 210 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 62.

Embodiment 211 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 63.

Embodiment 212 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 64.

Embodiment 213 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 65.

Embodiment 214 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 66.

Embodiment 215 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 67.

Embodiment 216 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 68.

Embodiment 217 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 69.

Embodiment 218 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 70.

Embodiment 219 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 71.

Embodiment 220 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 72.

Embodiment 221 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 73.

Embodiment 222 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 74.

Embodiment 223 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 75.

Embodiment 224 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 76.

Embodiment 225 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 77.

Embodiment 226 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 78.

Embodiment 227 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 79.

Embodiment 228 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 80.

Embodiment 229 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 81.

Embodiment 230 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 82.

Embodiment 231 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 83.

Embodiment 232 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 84.

Embodiment 233 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 85.

Embodiment 234 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 86.

Embodiment 235 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 87.

Embodiment 236 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 88.

Embodiment 237 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 89.

Embodiment 238 is the method, composition for use, or composition of anyone of embodiments 1-89, wherein the sequence selected from SEQ ID NOs:1-1491-149 is SEQ ID NO: 90.

Embodiment 239 is the method, composition for use, or composition of anyone of embodiments 1-89, wherein the sequence selected from SEQ ID NOs:1-1491-149 is SEQ ID NO: 91.

Embodiment 240 is the method, composition for use, or composition of anyone of embodiments 1-89, wherein the sequence selected from SEQ ID NOs:1-1491-149 is SEQ ID NO: 92.

Embodiment 241 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 93.

Embodiment 242 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 94.

Embodiment 243 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 95.

Embodiment 244 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 96.

Embodiment 245 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 97.

Embodiment 246 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 98.

Embodiment 247 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 99.

Embodiment 248 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 100.

Embodiment 249 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 101.

Embodiment 250 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 102.

Embodiment 251 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 103.

Embodiment 252 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 104.

Embodiment 253 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 105.

Embodiment 254 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 106.

Embodiment 255 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 107.

Embodiment 256 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 108.

Embodiment 257 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 109.

Embodiment 258 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 110.

Embodiment 259 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 111.

Embodiment 260 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 112.

Embodiment 261 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 113.

Embodiment 262 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 114.

Embodiment 263 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 115.

Embodiment 264 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 116.

Embodiment 265 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 117.

Embodiment 266 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 118.

Embodiment 267 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 119.

Embodiment 268 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 120.

Embodiment 269 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 121.

Embodiment 270 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 122.

Embodiment 271 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 123.

Embodiment 272 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 124.

Embodiment 273 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 125.

Embodiment 274 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 126.

Embodiment 275 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 127.

Embodiment 276 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 128.

Embodiment 277 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 129.

Embodiment 278 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 130.

Embodiment 279 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 131.

Embodiment 280 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 132.

Embodiment 281 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 133.

Embodiment 282 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 134.

Embodiment 283 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 135.

Embodiment 284 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 136.

Embodiment 285 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 137.

Embodiment 286 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 138.

Embodiment 287 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 139.

Embodiment 288 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 140.

Embodiment 289 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 141.

Embodiment 290 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 142.

Embodiment 291 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 143.

Embodiment 292 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 144.

Embodiment 293 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 145.

Embodiment 294 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 146.

Embodiment 295 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 147.

Embodiment 296 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 148.

Embodiment 297 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the composition comprising asequence selected from SEQ ID NOs: 1-149 is SEQ ID NO: 149.

Embodiment 298 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the guide sequence is selectedfrom SEQ ID NO: 310-386.

Embodiment 299 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the guide sequence is selectedfrom SEQ ID NO: 310-311, 313-326, 329-337, 339-342, 344-346, 348, 350,352-356, 361, 362, 364, 365, 366, 367, 369-374, 376-380, and 382-386.

Embodiment 300 is the method, composition for use, or composition of anyone of the preceding embodiments, wherein the guide sequence is selectedfrom SEQ ID NO: 310-386 is SEQ ID NO: 310.

Embodiment 301 is the method, composition for use, or composition of anyone of the preceding embodiments, comprising an sgRNAs comprising theguide sequence of any one of SEQ ID NOs: 1-149 and any one of theconserved portions of sgRNA of Table 4, optionally having themodification pattern of SEQ ID NO: 450 or any one of the modificationpatterns of Table 4, optionally wherein the sgRNA comprises a 5′ and 3′end modification.

Embodiment 302 is the method, composition, or composition for use of anyone of embodiments 1-301, wherein the composition is administered as asingle dose.

Embodiment 303 is the method, composition, or composition for use of anyone of embodiments 1-301, wherein the composition is administered onetime.

Embodiment 304 is the method, composition, or composition for use of anyone of embodiments 302 or 303, wherein the single dose or one timeadministration:

-   -   a. inducing a double-stranded break (DSB) within the KLKB1 gene        in a cell or subject; and/or    -   b. reducing expression of the KLKB1 gene in a cell or subject;        and/or    -   c. treating or preventing HAE in a subject; and/or    -   d. treating or preventing angioedema caused by or associated        with HAE in a subject; and/or    -   e. reducing serum and/or plasma bradykinin concentration in a        subject;    -   f. reducing bradykinin-mediated vasodilation;    -   g. treating or preventing bradykinin-mediated swelling and        angioedema; and/or    -   h. treating or preventing obstruction of the airway or        asphyxiation caused by swelling.

Embodiment 305 is the method or composition of embodiment 304, whereinthe single dose or one time administration achieves any one or more ofa)-h) for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks.

Embodiment 306 is the method or composition of embodiment 304, whereinthe single dose or one time administration achieves a durable effect.

Embodiment 307 is the method, composition, or composition for use of anyone of embodiments 1-306, further comprising achieving a durable effect.

Embodiment 308 is the method, composition, or composition for use ofembodiment 307, wherein the durable effect persists at least 1 month, atleast 3 months, at least 6 months, at least one year, or at least 5years.

Embodiment 309 is the method, composition, or composition for use of anyone of embodiments 1-308, wherein administration of the compositionresults in a therapeutically relevant reduction of kallikrein activity,total plasma kallikrein levels, prekallikrein and/or kallikrein levels,or bradykinin in serum and/or plasma.

Embodiment 310 is the method, composition, or composition for use of anyone of embodiments 1-309, wherein administration of the compositionresults in serum and/or plasma bradykinin levels within a therapeuticrange.

Embodiment 311 is the method, composition, or composition for use of anyone of the preceding embodiments, wherein administration of thecomposition results in serum and/or plasma bradykinin levels within 100,120, or 150% of normal range.

Embodiment 312 is use of a composition of any of the precedingcomposition embodiments for the preparation of a medicament for treatinga human subject having HAE.

Embodiment 313 is use of a composition of any of the precedingcomposition embodiments for the preparation of a medicament for treatingand preventing bradykinin production and accumulation,bradykinin-induced swelling, angioedema obstruction of the airway, orasphyxiation.

Embodiment 314 is use of a composition of any of the precedingcomposition embodiments for the preparation of a medicament for treatingor preventing angioedema caused by or associated with HAE.

Embodiment 315 is use of a composition of any of the precedingcomposition embodiments for the preparation of a medicament for reducingthe frequency of angioedema attacks.

Embodiment 316 is use of a composition of any of the precedingcomposition embodiments for the preparation of a medicament for reducingthe severity of angioedema attacks.

Embodiment 317 is use of a composition of any of the precedingcomposition embodiments for the preparation of a medicament for reducingthe frequency and/or severity of HAE attacks.

Embodiment 318 is use of a composition of any of the precedingcomposition embodiments for the preparation of a medicament forachieving remission of angioedema attacks.

Embodiment 319 is use of a composition of any of the precedingcomposition embodiments for the preparation of a medicament forachieving durable remission, e.g. maintained for at least 1 month, 2months, 4 months, 6 months, 1 year, 2 years, 5 years, 10 years or more.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D shows percent editing (indel frequency) detected at varioussites within the KLKB1 locus using guide RNAs in primary humanhepatocytes (PHH) (FIGS. 1A-1B) and primary cynomolgus hepatocytes (PCH)(FIGS. 1C-1D).

FIGS. 2A-2D show percent editing (indel frequency) of KLKB1 sgRNAs inPHH (FIGS. 2A-2B) and PCH (FIGS. 2C-2D).

FIGS. 3A-3E show percent editing (indel frequency) (FIG. 3A), secretedKLKB1 protein levels (FIG. 3B), and correlation plots (FIGS. 3C-E),after transfection of PHH with KLKB1-targeting guide RNAs in threedifferent PHH lots (HU8300, HU8284, and HU8296).

FIGS. 4A-4D show percent editing of the KLKB1 guides in primary humanhepatocytes (PHH) (FIGS. 4A-4B), and percent editing of the KLKB1 guidesin primary cynomolgus hepatocytes (PCH) (FIGS. 4C-4D).

FIGS. 5A-5J show dose response data for percent editing and secretedkallikrein for certain guide sequences in PHH (FIGS. 5A-5D) and PCH(FIGS. 5E-5H), and correlation plots of percent editing and secretedprotein in PHH and PCH (FIGS. 5I-5J).

FIGS. 6A-6D provide dose response curve data for indel frequency forcertain guide sequences in PHH (FIGS. 6A-6B) and PCH (FIGS. 6C-6D).

FIGS. 7A-7E show dose response curve data for indel frequency (FIGS. 7Aand 7B) and KLKB1 secretion (FIGS. 7C and 7D) for certain guidesequences in PHH (FIGS. 7A and 7C) and PCH (FIGS. 7B and 7D) and westernblot analysis to measure secreted protein (FIG. 7E).

FIG. 8A shows KLKB1 editing % for various modified sgRNAs in vivo in HuKLKB1 mice.

FIGS. 8B and 8C show KLKB1 protein levels measured using the ELISA andelectrochemiluminescence-based array respectively in Hu KLKB1 mice(Example 6).

FIG. 8D shows the fold change of KLKB1 mRNA levels for each sequence inHu KLKB1 mice.

FIGS. 9A-9D show levels of KLKB1 editing (FIG. 9A), serum KLKB1 protein(prekallikrein and kallikrein) (FIG. 9B), percent TSS (FIG. 9C) intreated mice, and the correlation of percent liver editing to percentKLKB1 protein (FIG. 9D).

FIG. 10 shows dose-dependent levels of KLKB1 gene editing, percentknockdown of KLKB1 mRNA, and plasma kallikrein in Hu KLKB1 mouse model.

FIG. 11A shows levels of KLKB1 gene editing and plasma kallikrein in adose response assay at after treatment with the indicated doses of sgRNAin Hu KLKB1 mouse model.

FIG. 11B shows levels of absorbance at 600 nm light to detect Evans blue(EB) dye from colon samples in a dose response vascular permeabilityassay in response to treatment with permeabilizing agents at aftertreatment with the indicated doses of sgRNA in Hu KLKB1 mouse model.

FIGS. 12A-12B show in vivo dose-dependent reductions in circulatingtotal kallikrein activity (FIG. 12A) and protein levels (FIG. 12B),respectively, after a single dose administration of CRISPR/Cas9components at 1.5 mg/kg, 3 mg/kg, or 6 mg/kg with G013901 in cynomolgusmonkeys.

FIGS. 13A-13B show in vivo reductions in circulating total kallikreinactivity (FIG. 13A) and protein levels (FIG. 13B), respectively, after asingle dose administration of CRISPR/Cas9 components at the indicateddosages with G012267 in cynomologous monkeys.

FIG. 14 labels the 10 conserved region YA sites in an exemplary sgRNAsequence (SEQ ID NO: 201) from 1 to 10. The numbers 25, 45, 50, 56, 64,67, and 83 indicate the position of the pyrimidine of YA sites 1, 5, 6,7, 8, 9, and 10 in an sgRNA with a guide region indicated as (N)x, e.g.,wherein x is optionally 20.

FIG. 15 shows an exemplary sgRNA (SEQ ID NO: 401; not all modificationsare shown) in a possible secondary structure with labels designatingindividual nucleotides of the conserved region of the sgRNA, includingthe lower stem, bulge, upper stem, nexus (the nucleotides of which canbe referred to as N1 through N18, respectively, in the 5′ to 3′direction), hairpin 1, and hairpin 2 regions. A nucleotide betweenhairpin 1 and hairpin 2 is labeled n. A guide region may be present onan sgRNA and is indicated in this figure as “(N)x” preceding theconserved region of the sgRNA.

DETAILED DESCRIPTION

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. While the invention is described in conjunction with theillustrated embodiments, it will be understood that they are notintended to limit the invention to those embodiments. On the contrary,the invention is intended to cover all alternatives, modifications, andequivalents, which may be included within the invention as defined bythe appended claims and included embodiments.

Before describing the present teachings in detail, it is to beunderstood that the disclosure is not limited to specific compositionsor process steps, as such may vary. It should be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. Thus, for example, reference to “a conjugate” includes aplurality of conjugates and reference to “a cell” includes a pluralityof cells and the like.

Numeric ranges are inclusive of the numbers defining the range. Measuredand measurable values are understood to be approximate, taking intoaccount significant digits and the error associated with themeasurement. Also, the use of “comprise”, “comprises”, “comprising”,“contain”, “contains”, “containing”, “include”, “includes”, and“including” are not intended to be limiting. It is to be understood thatboth the foregoing general description and detailed description areexemplary and explanatory only and are not restrictive of the teachings.

Unless specifically noted in the specification, embodiments in thespecification that recite “comprising” various components are alsocontemplated as “consisting of” or “consisting essentially of” therecited components; embodiments in the specification that recite“consisting of” various components are also contemplated as “comprising”or “consisting essentially of” the recited components; and embodimentsin the specification that recite “consisting essentially of” variouscomponents are also contemplated as “consisting of” or “comprising” therecited components (this interchangeability does not apply to the use ofthese terms in the claims).

The term “or” is used in an inclusive sense, i.e., equivalent to“and/or,” unless the context clearly indicates otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the desired subject matter inany way. In the event that any material incorporated by referencecontradicts any term defined in this specification or any other expresscontent of this specification, this specification controls. While thepresent teachings are described in conjunction with various embodiments,it is not intended that the present teachings be limited to suchembodiments. On the contrary, the present teachings encompass variousalternatives, modifications, and equivalents, as will be appreciated bythose of skill in the art.

I. Definitions

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

“Polynucleotide” and “nucleic acid” are used herein to refer to amultimeric compound comprising nucleosides or nucleoside analogs whichhave nitrogenous heterocyclic bases or base analogs linked togetheralong a backbone, including conventional RNA, DNA, mixed RNA-DNA, andpolymers that are analogs thereof. A nucleic acid “backbone” can be madeup of a variety of linkages, including one or more ofsugar-phosphodiester linkages, peptide-nucleic acid bonds (“peptidenucleic acids” or PNA; PCT No. WO 95/32305), phosphorothioate linkages,methylphosphonate linkages, or combinations thereof. Sugar moieties of anucleic acid can be ribose, deoxyribose, or similar compounds withsubstitutions, e.g., 2′ methoxy or 2′ halide substitutions. Nitrogenousbases can be conventional bases (A, G, C, T, U), analogs thereof (e.g.,modified uridines such as 5-methoxyuridine, pseudouridine, orN1-methylpseudouridine, or others); inosine; derivatives of purines orpyrimidines (e.g., N4-methyl deoxyguanosine, deaza- or aza-purines,deaza- or aza-pyrimidines, pyrimidine bases with substituent groups atthe 5 or 6 position (e.g., 5-methylcytosine), purine bases with asubstituent at the 2, 6, or 8 positions, 2-amino-6-methylaminopurine,O⁶-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines; U.S. Pat. No.5,378,825 and PCT No. WO 93/13121). For general discussion see TheBiochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11^(th) ed.,1992). Nucleic acids can include one or more “abasic” residues where thebackbone includes no nitrogenous base for position(s) of the polymer(U.S. Pat. No. 5,585,481). A nucleic acid can comprise only conventionalRNA or DNA sugars, bases and linkages, or can include both conventionalcomponents and substitutions (e.g., conventional bases with 2′ methoxylinkages, or polymers containing both conventional bases and one or morebase analogs). Nucleic acid includes “locked nucleic acid” (LNA), ananalogue containing one or more LNA nucleotide monomers with a bicyclicfuranose unit locked in an RNA mimicking sugar conformation, whichenhance hybridization affinity toward complementary RNA and DNAsequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). RNAand DNA have different sugar moieties and can differ by the presence ofuracil or analogs thereof in RNA and thymine or analogs thereof in DNA.

“Guide RNA”, “gRNA”, and simply “guide” are used herein interchangeablyto refer to the guide that directs an RNA-guided DNA binding agent to atarget DNA and can be either a crRNA (also known as CRISPR RNA), or thecombination of a crRNA and a trRNA (also known as tracrRNA). The crRNAand trRNA may be associated as a single RNA molecule (single guide RNA,sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). “GuideRNA” or “gRNA” refers to each type. The trRNA may be anaturally-occurring sequence, or a trRNA sequence with modifications orvariations compared to naturally-occurring sequences.

As used herein, a “guide sequence” refers to a sequence within a guideRNA that is complementary to a target sequence and functions to direct aguide RNA to a target sequence for binding or modification (e.g.,cleavage) by an RNA-guided DNA binding agent. A “guide sequence” mayalso be referred to as a “targeting sequence,” or a “spacer sequence.” Aguide sequence can be 20 base pairs in length, e.g., in the case ofStreptococcus pyogenes (i.e., Spy Cas9) and related Cas9homologs/orthologs. Shorter or longer sequences can also be used asguides, e.g., 15-, 16-, 17-, 18-, 19-, 21-, 22-, 23-, 24-, or25-nucleotides in length. For example, in some embodiments, the guidesequence comprises at least 17, 18, 19, or 20 contiguous nucleotides ofa sequence selected from SEQ ID NOs: 1-149. In some embodiments, thetarget sequence is in a gene or on a chromosome, for example, and iscomplementary to the guide sequence. In some embodiments, the degree ofcomplementarity or identity between a guide sequence and itscorresponding target sequence may be about 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100%. For example, in some embodiments, the guidesequence comprises a sequence with about 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% identity to at least 17, 18, 19, or 20 contiguousnucleotides of a sequence selected from SEQ ID NOs: 1-149. In someembodiments, the guide sequence and the target region may be 100%complementary or identical. In other embodiments, the guide sequence andthe target region may contain at least one mismatch. For example, theguide sequence and the target sequence may contain 1, 2, 3, or 4mismatches, where the total length of the target sequence is at least17, 18, 19, 20 or more base pairs. In some embodiments, the guidesequence and the target region may contain 1-4 mismatches where theguide sequence comprises at least 17, 18, 19, 20 or more nucleotides. Insome embodiments, the guide sequence and the target region may contain1, 2, 3, or 4 mismatches where the guide sequence comprises 20nucleotides.

Target sequences for RNA-guided DNA binding agents include both thepositive and negative strands of genomic DNA (i.e., the sequence givenand the sequence's reverse compliment), as a nucleic acid substrate foran RNA-guided DNA binding agent is a double stranded nucleic acid.Accordingly, where a guide sequence is said to be “complementary to atarget sequence”, it is to be understood that the guide sequence maydirect a guide RNA to bind to the reverse complement of a targetsequence. Thus, in some embodiments, where the guide sequence binds thereverse complement of a target sequence, the guide sequence is identicalto certain nucleotides of the target sequence (e.g., the target sequencenot including the PAM) except for the substitution of U for T in theguide sequence.

As used herein, an “RNA-guided DNA binding agent” means a polypeptide orcomplex of polypeptides having RNA and DNA binding activity, or aDNA-binding subunit of such a complex, wherein the DNA binding activityis sequence-specific and depends on the sequence of the RNA. ExemplaryRNA-guided DNA binding agents include Cas cleavases/nickases andinactivated forms thereof (“dCas DNA binding agents”). “Cas nuclease”,also called “Cas protein” as used herein, encompasses Cas cleavases, Casnickases, and dCas DNA binding agents. Cas cleavases/nickases and dCasDNA binding agents include a Csm or Cmr complex of a type III CRISPRsystem, the Cas10, Csm1, or Cmr2 subunit thereof, a Cascade complex of atype I CRISPR system, the Cas3 subunit thereof, and Class 2 Casnucleases. As used herein, a “Class 2 Cas nuclease” is a single-chainpolypeptide with RNA-guided DNA binding activity. Class 2 Cas nucleasesinclude Class 2 Cas cleavases/nickases (e.g., H840A, D10A, or N863Avariants), which further have RNA-guided DNA cleavases or nickaseactivity, and Class 2 dCas DNA binding agents, in which cleavase/nickaseactivity is inactivated. Class 2 Cas nucleases include, for example,Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926Avariants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants),eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1)(e.g., K848A, K1003A, R1060A variants) proteins and modificationsthereof. Cpf1 protein, Zetsche et al., Cell, 163: 1-13 (2015), ishomologous to Cas9, and contains a RuvC-like nuclease domain. Cpf1sequences of Zetsche are incorporated by reference in their entirety.See, e.g., Zetsche, Tables Si and S3. See, e.g., Makarova et al., NatRev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell.60:385-397 (2015).

As used herein, “ribonucleoprotein” (RNP) or “RNP complex” refers to aguide RNA together with an RNA-guided DNA binding agent, such as a Casnuclease, e.g., a Cas cleavase, Cas nickase, or dCas DNA binding agent(e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guidedDNA binding agent such as Cas9 to a target sequence, and the guide RNAhybridizes with and the agent binds to the target sequence; in caseswhere the agent is a cleavase or nickase, binding can be followed bycleaving or nicking.

As used herein, a first sequence is considered to “comprise a sequencewith at least X % identity to” a second sequence if an alignment of thefirst sequence to the second sequence shows that X % or more of thepositions of the second sequence in its entirety are matched by thefirst sequence. For example, the sequence AAGA comprises a sequence with100% identity to the sequence AAG because an alignment would give 100%identity in that there are matches to all three positions of the secondsequence. The differences between RNA and DNA (generally the exchange ofuridine for thymidine or vice versa) and the presence of nucleosideanalogs such as modified uridines do not contribute to differences inidentity or complementarity among polynucleotides as long as therelevant nucleotides (such as thymidine, uridine, or modified uridine)have the same complement (e.g., adenosine for all of thymidine, uridine,or modified uridine; another example is cytosine and 5-methylcytosine,both of which have guanosine or modified guanosine as a complement).Thus, for example, the sequence 5′-AXG where X is any modified uridine,such as pseudouridine, N1-methyl pseudouridine, or 5-methoxyuridine, isconsidered 100% identical to AUG in that both are perfectlycomplementary to the same sequence (5′-CAU). Exemplary alignmentalgorithms are the Smith-Waterman and Needleman-Wunsch algorithms, whichare well-known in the art. One skilled in the art will understand whatchoice of algorithm and parameter settings are appropriate for a givenpair of sequences to be aligned; for sequences of generally similarlength and expected identity >50% for amino acids or >75% fornucleotides, the Needleman-Wunsch algorithm with default settings of theNeedleman-Wunsch algorithm interface provided by the EBI at thewww.ebi.ac.uk web server is generally appropriate.

1 “mRNA” is used herein to refer to a polynucleotide and comprises anopen reading frame that can be translated into a polypeptide (i.e., canserve as a substrate for translation by a ribosome and amino-acylatedtRNAs). mRNA can comprise a phosphate-sugar backbone including riboseresidues or analogs thereof, e.g., 2′-methoxy ribose residues. In someembodiments, the sugars of an mRNA phosphate-sugar backbone consistessentially of ribose residues, 2′-methoxy ribose residues, or acombination thereof.

Guide sequences useful in the guide RNA compositions and methodsdescribed herein are shown in Table 1 or Table 2 and throughout theapplication.

As used herein, “indels” refer to insertion/deletion mutationsconsisting of a number of nucleotides that are either inserted ordeleted at the site of double-stranded breaks (DSBs) in a target nucleicacid.

As used herein, “knockdown” refers to a decrease in expression of aparticular gene product (e.g., protein, mRNA, or both). Knockdown of aprotein can be measured by detecting total cellular amount of theprotein from a sample, such as a tissue, fluid, or cell population ofinterest. It can also be measured by measuring a surrogate, marker, oractivity for the protein. Methods for measuring knockdown of mRNA areknown and include sequencing of mRNA isolated from a sample of interest.In some embodiments, “knockdown” may refer to some loss of expression ofa particular gene product, for example a decrease in the amount of mRNAtranscribed or a decrease in the amount of protein expressed by apopulation of cells (including in vivo populations such as those foundin tissues).

As used herein, “knockout” refers to a loss of expression from aparticular gene or of a particular protein in a cell. Knockout can bemeasured either by detecting total cellular amount of a protein in acell, a tissue or a population of cells. In some embodiments, themethods of the invention “knockout” KLKB1 in one or more samples, e.g.,serum, plasma, tissue, or cells (e.g., in a population of cellsincluding in vivo populations such as those found in tissues). In someembodiments, a knockout is not the formation of mutant KLKB1 protein,for example, created by indels, but rather the complete loss ofexpression of KLKB1 protein in a cell. As used herein, “KLKB1” generallyrefers to prekallikrein, which is the gene product of a KLKB1 gene.Prekallikrein is processed to plasma kallikrein (pKal), and antibodiescan detect pKal, prekallikrein, or both. The human wild-type KLKB1sequence is available at NCBI Gene ID: 3818; Ensembl: ENSG00000164344.“PKK,” “PPK,” “KLK3,” and “PKKD” are gene synonyms. The human KLKB1transcript is available at Ensembl: ENST00000264690, and the cynomolguswild-type KLKB1 sequence is available at Ensembl: ENSMFAT00000002355.

“Hereditary Angioedema” (HAE) is an inflammatory disorder characterizedby recurrent episodes of severe swelling (angioedema), due toinactivating mutations of the SERPING1 gene, which encodes the C1esterase inhibitor protein (C1-INH). C1-INH blocks the activity ofcertain proteins that promote inflammation (e.g., in Kinin system).Deficient levels of C1-INH leads to unchecked Factor XII (FXII) and highlevel of activation of kallikrein (pKal, processed from KLKB1 protein(prekallikrein)). Kallikrein cleaves high-molecular weight kininogen(HMWK) to release bradykinin, a peptide that impacts vascularpermeability. Excessive amount of bradykinin in the blood leads to thefluid leakage through the walls of blood vessels into body tissues,causing swelling seen in individuals with HAE. Thus, in someembodiments, methods for decreasing KLKB1 activity are provided, whereinonce reduced, bradykinin production is decreased and swelling attacksare reduced. Protein levels of prekallikrein/kallikrein, HMWK and itscleavage products, and surrogate labeled substrates of HMWK may bemeasured to assess efficacy of KLKB1 knockout.

As used herein, a “target sequence” refers to a sequence of nucleic acidin a target gene that has complementarity to the guide sequence of thegRNA. The interaction of the target sequence and the guide sequencedirects an RNA-guided DNA binding agent to bind, and potentially nick orcleave (depending on the activity of the agent), within the targetsequence.

As used herein, a “YA site” refers to a 5′-pyrimidine-adenine-3′dinucleotide. A “conserved region YA site” is present in the conservedregion of an sgRNA. A “guide region YA site” is present in the guideregion of an sgRNA. An unmodified YA site in an sgRNA may be susceptibleto cleavage by RNase-A like endonucleases, e.g., RNase A. In someembodiments, an sgRNA comprises about 10 YA sites in its conservedregion. In some embodiments, an sgRNA comprises 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 YA sites in its conserved region. Exemplary conserved region YAsites are indicated in FIG. 14 (SEQ ID NO: 201), in relation to an sgRNAstructure (FIG. 15 ). Exemplary guide region YA sites are not shown inFIG. 14 , as the guide region may be any sequence, including any numberof YA sites. In some embodiments, an sgRNA comprises 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 of the YA sites indicated in FIG. 14 . In someembodiments, an sgRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 YAsites at the following positions or a subset thereof: LS5-LS6; US3-US4;US9-US10; US12-B3; LS7-LS8; LS12-N1; N6-N7; N14-N15; N17-N18; and H2-2to H2-3. In some embodiments, a YA site comprises a modification,meaning that at least one nucleotide of the YA site is modified. In someembodiments, the pyrimidine (also called the pyrimidine position) of theYA site comprises a modification (which includes a modification alteringthe internucleoside linkage immediately 3′ of the sugar of thepyrimidine). In some embodiments, the adenine (also called the adenineposition) of the YA site comprises a modification (which includes amodification altering the internucleoside linkage immediately 3′ of thesugar of the adenine). In some embodiments, the pyrimidine position andthe adenine position of the YA site comprise modifications.

As used herein, “treatment” refers to any administration or applicationof a therapeutic for disease or disorder in a subject, and includesinhibiting the disease, arresting its development, relieving one or moresymptoms of the disease, curing the disease, or preventing reoccurrenceof one or more symptoms of the disease. For example, treatment of HAEmay comprise alleviating symptoms of HAE.

The term “therapeutically relevant reduction of KLKB1 activity,” canmean a greater than about 60% reduction of plasma KLKB1 activity ascompared to baseline. See, Banerji et al., N Engl J Med, 2017,376:717-728; Ferrone et al., Nucleic Acid Therapeutics, 2019, 82-917.KLKB1 activity is often measured as total kallikrein activity, in whichprekallikrein is converted to kallikrein in a sample and totalkallikrein activity is measured for the sample. In some instances, arange of KLKB1 activity reduction can mean about 60-80% reduction ofplasma KLKB1 activity as compared to baseline. To calculate reduction ofan analyte in a subject, a basal value can be obtained by collecting apretreatment sample from the subject. In some instances, the sample is aserum sample. In certain aspects, the target KLKB1 activity reduction isabout a 60% reduction in total kallikrein (prekallikrein and plasmakallikrein) activity as compared to baseline. For example, achievingKLKB1 activity levels within a therapeutic range can mean reducing totalkallikrein by about >60% from baseline. In some embodiments, a “normalkallikrein level” or a “normal kallikrein range” is reduced. In someembodiments, a therapeutically relevant reduction of kallikrein activityachieves levels of about 0-60%, 0-50%, 0-40%, 0-30%, 0-25%, 0-20%,0-15%, 0-10% of a basal value for the subject, or 10-60%, 10-50%,10-40%, 10-30%, 10-20%, or 20-60%, 20-50%, 20-40%, or 20-30%%, of normalkallikrein activity level. KLKB1 activity can be measured by assaysknown in the field, including assays described herein.

The term “target KLKB1 protein reduction,” as used herein, means thetarget level of pKal as compared to baseline. KLKB1 protein levels canbe measured by assays known in the field such as ELISA or western blotassays, as described herein. Total KLKB1 protein can be measured with anantibody that detects both prekallikrein and kallikrein and/or afterconverting prekallikrein to kallikrein in a sample. In some instances,the sample is a serum sample. In certain aspects, the target KLKB1protein reduction is about a 60% reduction in total kallikrein(prekallikrein and plasma kallikrein) as compared to baseline. In someembodiments, a therapeutically relevant reduction of total kallikreinprotein achieves levels of about 0-60%, 0-50%, 0-40%, 0-30%, 0-25%,0-20%, 0-15%, 0-10% of a basal value for the subject, or 10-60%, 10-50%,10-40%, 10-30%, 10-20%, or 20-60%, 20-50%, 20-40%, or 20-30%%, of normaltotal kallikrein protein level.

Circulating plasma cHMWK levels below about 30% total HMWK wereassociated with decreases in HAE attacks in patients treated withlanadelumab (See Banerji, et al, 2017). In this same study, healthycontrols had plasma levels of cHMWK around 8.3% total HMWK. In anotherstudy, Suffriti and colleagues found cHMWK plasma levels of an averageof about 34.8% in normal controls, about 41.4% in HAE patients inremission and about 58.1% in HAE patients during an attack (Suffritti,et al. Clin Exp Allergy 2014; 44:1503-14). Therapeutic treatment cantarget a ratio of circulating plasma cHMWK to total HMWK of less thanabout 60%. In some embodiments the ratio of cHMWK to HMWK is less thanabout 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, or more.

The term “about” or “approximately” means an acceptable error for aparticular value as determined by one of ordinary skill in the art,which depends in part on how the value is measured or determined.

II. Compositions

A. Compositions Comprising Guide RNA (gRNAs)

Provided herein are compositions useful for inducing a double-strandedbreak (DSB), single-strand break, and/or site-specific binding thatresults in nucleic acid modification within the KLKB1 gene, e.g., usinga guide RNA with an RNA-guided DNA binding agent (e.g., a CRISPR/Cassystem). The compositions may be administered to subjects having orsuspected of having HAE. The compositions may be administered tosubjects having increased serum and/or plasma bradykinin concentrationas measured, for example, by a decrease in prekallikrein protein levelsin the plasma or serum, by a decrease in total kallikrein (prekallikreinand pKal) protein levels in plasma or serum, by a decrease in theproportion of circulating cleaved HMWK (cHMWK), or by a decrease in theproportion of cHMWK in citrated plasma. The compositions may beadministered to subjects having increased serum and/or plasmaprekallikrein and/or kallikrein concentration. The compositions may beadministered to subjects having increased serum and/or plasma totalkallikrein concentration. The compositions may be administered tosubjects having increased serum and/or plasma kallikrein activity. Guidesequences targeting the KLKB1 gene are shown in Table 1 at SEQ ID NOs:1-149.

Each of the guide sequences shown in Table 1 at SEQ ID NOs: 1-149 mayfurther comprise additional nucleotides to form a crRNA, e.g., with thefollowing exemplary nucleotide sequence following the guide sequence atits 3′ end: GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 167) in 5′ to 3′orientation. In the case of a sgRNA, the above guide sequences mayfurther comprise additional nucleotides to form a sgRNA, e.g., with thefollowing exemplary nucleotide sequence following the 3′ end of theguide sequence: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 171) orGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 172, which is SEQ ID NO: 171without the four terminal U's) in 5′ to 3′ orientation. In someembodiments, the four terminal U's of SEQ ID NO: 171 are not present. Insome embodiments, only 1, 2, or 3 of the four terminal U's of SEQ ID NO:171 are present.

In some embodiments, the sgRNA comprises any one of the guide sequencesof SEQ ID Nos: 1-149 and additional nucleotides to form a crRNA, e.g.,with the following exemplary nucleotide sequence following the guidesequence at its 3′ end:GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 170) orGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GGCACCGAGUCGGUGC(SEQ ID NO: 173) in 5′ to 3′ orientation. SEQ ID NO: 173 lacks 8nucleotides with reference to a wild-type guide RNA conserved sequence:GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 172).

In some embodiments, KLKB1 short-single guide RNAs (KLKB1 short-sgRNAs)are provided comprising a guide sequence as described herein and a“conserved portion of an sgRNA” comprising a hairpin region, wherein thehairpin region lacks at least 5-10 nucleotides or 6-10 nucleotides. Incertain embodiments, a hairpin region of the KLKB1 short-single guideRNAs lacks 5-10 nucleotides with reference to the conserved portion ofan sgRNA, e.g. nucleotides H1-1 to H2-15 in Table 3B and FIG. 15 . Incertain embodiments, a hairpin 1 region of the KLKB1 short-single guideRNAs lacks 5-10 nucleotides with reference to the conserved portion ofan sgRNA, e.g. nucleotides H1-1 to H1-12 in Table 3B and FIG. 15 . See,e.g., WO2019/237069, the contents of which is hereby incorporated byreference in its entirety, for example, at claims 1-15.

An exemplary “conserved portion of an sgRNA” is shown in Table 3A (seealso FIG. 15 ), which shows a “conserved region” of a S. pyogenes Cas9(“spyCas9” (also referred to as “spCas9”)) sgRNA. The first row showsthe numbering of the nucleotides, the second row shows the sequence (SEQID NO: 500); and the third row shows “domains.” Briner A E et al.,Molecular Cell 56:333-339 (2014) describes functional domains of sgRNAs,referred to herein as “domains”, including the “spacer” domainresponsible for targeting, the “lower stem”, the “bulge”, “upper stem”(which may include a tetraloop), the “nexus”, and the “hairpin 1” and“hairpin 2” domains. See, Briner et al. at page 334, FIG. 1A.

Table 3B provides a schematic of the domains of an sgRNA as used herein.In Table 3B, the “n” between regions represents a variable number ofnucleotides, for example, from 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or more. In some embodiments, nequals 0. In some embodiments, n equals 1.

In some embodiments, the KLKB1 sgRNA is from S. pyogenes Cas9(“spyCas9”) or a spyCas9 equivalent. In some embodiments, the sgRNA isnot from S. pyogenes (“non-spyCas9”). In some embodiments, the 5-10nucleotides or 6-10 nucleotides are consecutive.

In some embodiments, a KLKB1 short-sgRNA lacks at least nucleotides54-58 (AAAAA) of the conserved portion of a S. pyogenes Cas9 (“spyCas9”)sgRNA, as shown in Table 3A. In some embodiments, a KLKB1 short-sgRNA isa non-spyCas9 sgRNA that lacks at least nucleotides corresponding tonucleotides 54-58 (AAAAA) of the conserved portion of a spyCas9 asdetermined, for example, by pairwise or structural alignment. In someembodiments, the non-spyCas9 sgRNA is Staphylococcus aureus Cas9(“saCas9”) sgRNA.

In some embodiments, a KLKB1 short-sgRNA lacks at least nucleotides54-61 (AAAAAGUG) of the conserved portion of a spyCas9 sgRNA. In someembodiments, a KLKB1 short-sgRNA lacks at least nucleotides 53-60(GAAAAAGU) of the conserved portion of a spyCas9 sgRNA. In someembodiments, a KLKB1 short-sgRNA lacks 4, 5, 6, 7, or 8 nucleotides ofnucleotides 53-60 (GAAAAAGU) or nucleotides 54-61 (AAAAAGUG) of theconserved portion of a spyCas9 sgRNA, or the corresponding nucleotidesof the conserved portion of a non-spyCas9 sgRNA as determined, forexample, by pairwise or structural alignment.

TABLE 1 Human KLKB1 targeted guide sequence, chromosomal coordinates,and human single guide RNAs and dual guide RNAs, andsurrogate cynomolgus (cyno) monkey single guides Cyno SEQ ID guide NO:Exemplary Genomic human guide human human cyno SEQ ID (human)Coordinates (hg38) sequence sgRNA dgRNA sgRNA NO:   1chr4:186228230-186228252 ACAGGAAACUGUAGCAAACA G012253 CR005916 NA NA   2chr4:186228248-186228270 AUAGAUAAUUCACUUACCAC G012254 CR005917 NA NA   3chr4:186232154-186232176 UACAUCCCCACCUCUGAAGA G012255 CR005918 NA NA   4chr4:186251256-186251278 UCUUGAGGAGUAGAGGAACU G012256 CR005922 NA NA   5chr4:186251308-186251330 ACCAGGUAAAGUUCUUUUGC G012257 CR005924 NA NA   6chr4:186251489-186251511 GGGUAAAUUUUAGAAUGGCA G012258 CR005925 NA NA   7chr4:186251504-186251526 AUUUACCCGGGAGUUGACUU G012259 CR005928 NA NA   8chr4:186251507-186251529 UACCCGGGAGUUGACUUUGG G012260 CR005929 NA NA   9chr4:186251828-186251850 UCUUUGAGAUUGUGUAACAC G012261 CR005931 NA NA  10chr4:186251829-186251851 CUUUGAGAUUGUGUAACACU G012262 CR005932 NA NA  11chr4:186251830-186251852 UUUGAGAUUGUGUAACACUG G012263 CR005933 NA NA  12chr4:186254748-186254770 UACAUACCAGUGUAAUUCAA G012264 CR005943 NA NA  13chr4:186251784-186251806 CUCCAACUAGGAUUGCGUAU G012265 CR005949 G013933373  14 chr4:186251792-186251814 AGGAUUGCGUAUGGGACACA G012266 CR005951G013904 344  15 chr4:186251793-186251815 GGAUUGCGUAUGGGACACAA G012267CR005952 G013901 341  16 chr4:186238297-186238319 GUUACUCAGCACCUUUAUAGG012268 CR005956 G013945 385  17 chr4:186238263-186238285UGCCUAUUAAAGUACAGUCC G012269 CR005959 NA NA  18 chr4:186251772-186251794CUAUGGAUGGUUCUCCAACU G012270 CR005960 G013922 362  19chr4:186254601-186254623 GAUGUUUGGCGCAUCUAUAG G012271 CR005963 G013921361  20 chr4:186254592-186254614 AUGCGCCAAACAUCCUGCAG G012272 CR005970G013885 325  21 chr4:186236785-186236807 CUCCUUUAUAAAUGUCUCGA G012273CR005979 G013905 345  22 chr4:186236863-186236885 UGUUACUGGUGCACCUUUUUG012274 CR005982 NA NA  23 chr4:186254593-186254615 GAUGCGCCAAACAUCCUGCAG012275 CR005983 G013876 316  24 chr4:186232192-186232214AUCUGGCAGUAUUGGGCAUU G012276 CR005992 G013915 355  25chr4:186236893-186236915 GCGUGGCAUAUGAAAAAAAC G012277 CR005994 NA NA  26chr4:186236798-186236820 UAUAAAGGAGUUGAUAUGAG G012278 CR005995 G013913353  27 chr4:186236938-186236960 ACACCUUGAAUUGUACUCAC G012279 CR005998NA NA  28 chr4:186232214-186232236 UGAGGUGCACAUUCCACCCA G012280 NA NA NA 29 chr4:186232190-186232212 CUGGCAGUAUUGGGCAUUUG G012281 NA NA NA  30chr4:186232148-186232170 AAAACGCCUUCUUCAGAGGU G012282 NA NA NA  31chr4:186232227-186232249 UGAAUAGCAAACACCUUGGG G012283 NA NA NA  32chr4:186236821-186236843 AGUCAAUUUUAAUGUGUCUA G012284 NA NA NA  33chr4:186236850-186236872 GUGUUGAAGAAUGCCAAAAA G012285 NA NA NA  34chr4:186236910-186236932 UGCCUUGUGAAAUGUUUGCG G012286 NA NA NA  35chr4:186250265-186250287 GCAUCUUGCGUUCUCAGAUG G012287 NA G013927 367  36chr4:186250276-186250298 UCUCAGAUGUGGAUGUUGCC G012288 NA NA NA  37chr4:186250306-186250328 CUCCAGAUGCUUUUGUGUGU G012289 NA NA NA  38chr4:186251325-186251347 UAUUAUCAAAUCACAUUACC G012290 NA NA NA  39chr4:186251271-186251293 CCAGAUAUGGUGUUUUCUUG G012291 NA NA NA  40chr4:186251300-186251322 AAGUUCUUUUGCAGGUUAAA G012292 NA NA NA  41chr4:186251620-186251642 UUUACUCCCAGAAGACUGUA G012293 NA NA NA  42chr4:186251492-186251514 UGCCAUUCUAAAAUUUACCC G012294 NA NA NA  43chr4:186251572-186251594 UCAUCUUUGUGCAAGUCUCU G012295 NA NA NA  44chr4:186251510-186251532 UCUCCUCCAAAGUCAACUCC G012296 NA NA NA  45chr4:186252049-186252071 GGAGGAACAAACUCUUCUUG G012297 NA NA NA  46chr4:186252098-186252120 AGGUGAAGCUGACAGCUCAG G012298 NA NA NA  47chr4:186256046-186256068 CCAUCCGGUUACCCAACAGU G012299 NA G013931 371  48chr4:186256042-186256064 UAUACCAACUGUUGGGUAAC G012300 NA G012300 NA  49chr4:186256034-186256056 GCACAAUUUAUACCAACUGU G012301 NA NA NA  50chr4:186256059-186256081 AACCGGAUGGGGCUUCUCGA G012302 NA G013932 372  51chr4:186256047-186256069 CAACUGUUGGGUAACCGGAU G012303 NA G013882 322  52chr4:186256035-186256057 CACAAUUUAUACCAACUGUU G012304 NA G012304 NA  53chr4:186256046-186256068 CCAACUGUUGGGUAACCGGA G012305 NA G013924 364  54chr4:186256061-186256083 CUCCUUCGAGAAGCCCCAUC G012306 NA NA NA  55chr4:186256048-186256070 AACUGUUGGGUAACCGGAUG G012307 NA G013914 354  56chr4:186256003-186256025 CCAAUAUGCCUACCUUCCAA G012308 NA NA NA  57chr4:186256015-186256037 GUGCUUGUGUCACCUUUGGA G012309 NA G013900 340  58chr4:186256011-186256033 UUGUGUCACCUUUGGAAGGU G012310 NA NA NA  59chr4:186256019-186256041 AAUUGUGCUUGUGUCACCUU G012311 NA NA NA  60chr4:186255996-186256018 AAGGUAGGCAUAUUGGUUUU G012312 NA NA NA  61chr4:186257312-186257334 ACCCAACGGAUGGUCUGUGC G012313 NA NA NA  62chr4:186257314-186257336 AGCCAGCACAGACCAUCCGU G012314 NA NA NA  63chr4:186257302-186257324 UUAUAAAAUAACCCAACGGA G012315 NA G012315 NA  64chr4:186257326-186257348 CUGUGCUGGCUAUAAAGAAG G012316 NA NA NA  65chr4:186257261-186257283 CAUUCUUCAUUUGUUACCAA G012317 NA NA NA  66chr4:186257284-186257306 UAUAAUCUUGAUAUCUUUUC G012318 NA NA NA  67chr4:186257313-186257335 GCCAGCACAGACCAUCCGUU G012319 NA NA NA  68chr4:186257324-186257346 GUCUGUGCUGGCUAUAAAGA G012320 NA G012320 NA  69chr4:186257325-186257347 UCUGUGCUGGCUAUAAAGAA G012321 NA NA NA  70chr4:186258130-186258152 GUCCAUGUACUCAGCGACUU G012322 NA G012322 NA  71chr4:186258128-186258150 CACCAAAGUCGCUGAGUACA G012323 NA G012323 NA  72chr4:186258050-186258072 ACACAAUGGAAUGUGGCGUU G012324 NA G012324 NA  73chr4:186258068-186258090 UUUGGUGGGCAUCACCAGCU G012325 NA G012325 NA  74chr4:186258204-186258226 CUCUGGACUGCUUCUCAUGC G012326 NA NA NA  75chr4:186258133-186258155 AAGUCGCUGAGUACAUGGAC G012327 NA G012327 NA  76chr4:186258089-186258111 GGGUGAAGGCUGUGCCCGCA G012328 NA G013895 335  77chr4:186258054-186258076 AAUGGAAUGUGGCGUUUGGU G012329 NA G012329 NA  78chr4:186258037-186258059 UCCAUUGUGUUUGCAAACUA G012330 NA G013942 382  79chr4:186258067-186258089 GUUUGGUGGGCAUCACCAGC G012331 NA NA NA  80chr4:186258043-186258065 UUUGCAAACACAAUGGAAUG G012332 NA G013916 356  81chr4:186258103-186258125 GACACCAGGUUGCUCCCUGC G012333 NA NA NA  82chr4:186258009-186258031 ACUGUGACUCAGGGAGAUUC G012334 NA G013943 383  83chr4:186258099-186258121 UGUGCCCGCAGGGAGCAACC G012335 NA NA NA  84chr4:186258036-186258058 CCAUUGUGUUUGCAAACUAA G012336 NA G013929 369  85chr4:186258088-186258110 GGGGUGAAGGCUGUGCCCGC G012337 NA NA NA  86chr4:186258117-186258139 GCGACUUUGGUGUAGACACC G012338 NA NA NA  87chr4:186258036-186258058 CCCUUAGUUUGCAAACACAA G012339 NA NA NA  88chr4:186258053-186258075 CAAUGGAAUGUGGCGUUUGG G012340 NA G012340 NA  89chr4:186232230-186232252 AACUGAAUAGCAAACACCUU NA CR005919 NA NA  90chr4:186238351-186238373 ACAAUUACCAAUUUCUGAAA NA CR005920 NA NA  91chr4:186238352-186238374 UACAAUUACCAAUUUCUGAA NA CR005921 NA NA  92chr4:186251263-186251285 GGUGUUUUCUUGAGGAGUAG NA CR005923 NA NA  93chr4:186251490-186251512 CGGGUAAAUUUUAGAAUGGC NA CR005926 G013884 324 94 chr4:186251494-186251516 CUCCCGGGUAAAUUUUAGAA NA CR005927 G013925365  95 chr4:186251801-186251823 UAUGGGACACAAGGGAGCUC NA CR005930 NA NA 96 chr4:186252047-186252069 UUGGAGGAACAAACUCUUCU NA CR005934 G013912352  97 chr4:186252048-186252070 UGGAGGAACAAACUCUUCUU NA CR005935 NA NA 98 chr4:186252056-186252078 CAAACUCUUCUUGGGGAGAG NA CR005936 NA NA  99chr4:186252123-186252145 CUAUGAGUGACCCUCCACAC NA CR005937 G013886 326100 chr4:186252124-186252146 CUGUGUGGAGGGUCACUCAU NA CR005938 G013938378 101 chr4:186252134-186252156 GGUCACUCAUAGGACACCAG NA CR005939G013946 386 102 chr4:186252135-186252157 GUCACUCAUAGGACACCAGU NACR005940 G013896 336 103 chr4:186252163-186252185 ACUGCUGCCCACUGCUUUGANA CR005941 NA NA 104 chr4:186252171-186252193 ACACUUACCCAUCAAAGCAG NACR005942 G013902 342 105 chr4:186238286-186238308 AGGAACACCUACCGCUAUAANA CR005944 G013871 311 106 chr4:186238265-186238287CUCCGGGACUGUACUUUAAU NA CR005945 G013889 329 107chr4:186251786-186251808 GUCCCAUACGCAAUCCUAGU NA CR005946 G013890 330108 chr4:186238293-186238315 CUCAGCACCUUUAUAGCGGU NA CR005947 G013892332 109 chr4:186238282-186238304 UAUAGCGGUAGGUGUUCCUC NA CR005948G013874 314 110 chr4:186238266-186238288 CUAUUAAAGUACAGUCCCGG NACR005950 G013875 315 111 chr4:186238308-186238330 GUGCUGAGUAACGUGGAAUCNA CR005953 G013883 323 112 chr4:186238301-186238323UAUAAAGGUGCUGAGUAACG NA CR005954 G013878 318 113chr4:186251783-186251805 UCUCCAACUAGGAUUGCGUA NA CR005955 G013908 348114 chr4:186238281-186238303 AUAGCGGUAGGUGUUCCUCC NA CR005957 G013873313 115 chr4:186233989-186234011 CUGCCAAAAGUACAUCGAAC NA CR005958G013877 317 116 chr4:186238345-186238367 ACCAAUUUCUGAAAGGGCAC NACR005961 NA NA 117 chr4:186251755-186251777 GUGUUUCUUAAGAUUAUCUA NACR005962 NA NA 118 chr4:186238344-186238366 CCAAUUUCUGAAAGGGCACA NACR005964 NA NA 119 chr4:186251759-186251781 UUCUUAAGAUUAUCUAUGGA NACR005965 G013940 380 120 chr4:186233988-186234010 CUGUUCGAUGUACUUUUGGCNA CR005966 NA NA 121 chr4:186233987-186234009 UGUUCGAUGUACUUUUGGCA NACR005967 G013880 320 122 chr4:186232209-186232231 GGUGGAAUGUGCACCUCAUCNA CR005968 G013939 379 123 chr4:186250308-186250330GUCCGACACACAAAAGCAUC NA CR005969 G013894 334 124chr4:186236877-186236899 AAACUGGCAGCGAAUGUUAC NA CR005971 G013930 370125 chr4:186236908-186236930 UGCCACGCAAACAUUUCACA NA CR005972 NA NA 126chr4:186233992-186234014 GCACCUGUUCGAUGUACUUU NA CR005973 G013870 310127 chr4:186254594-186254616 AGAUGCGCCAAACAUCCUGC NA CR005974 NA NA 128chr4:186232199-186232221 GCACCUCAUCUGGCAGUAUU NA CR005975 NA NA 129chr4:186250262-186250284 CAUCUGAGAACGCAAGAUGC NA CR005976 G013934 374130 chr4:186232196-186232218 AUGCCCAAUACUGCCAGAUG NA CR005977 NA NA 131chr4:186232200-186232222 UGCACCUCAUCUGGCAGUAU NA CR005978 G013944 384132 chr4:186232258-186232280 AUGUCAUUGAUUGAACUUGC NA CR005980 G013936376 133 chr4:186252031-186252053 ACAAGCACACGCAUUGUUGG NA CR005981G013893 333 134 chr4:186254723-186254745 UAUCGCCUUGAUAAAACUCC NACR005984 G013926 366 135 chr4:186251271-186251293 CCUCAAGAAAACACCAUAUCNA CR005985 G013906 346 136 chr4:186232149-186232171AAACGCCUUCUUCAGAGGUG NA CR005986 NA NA 137 chr4:186252028-186252050AAAACAAGCACACGCAUUGU NA CR005987 G013891 331 138chr4:186234001-186234023 CAUCGAACAGGUGCAGUUUC NA CR005988 G013879 319139 chr4:186254587-186254609 GGCUUCCCCUGCAGGAUGUU NA CR005989 G013881321 140 chr4:186234029-186234051 UUGAUGACCACAUUGCUUCA NA CR005990G013937 377 141 chr4:186254728-186254750 AGGAGCCUGGAGUUUUAUCA NACR005991 NA NA 142 chr4:186236783-186236805 UGCCAUCGAGACAUUUAUAA NACR005993 G013899 339 143 chr4:186232260-186232282 AGCAAGUUCAAUCAAUGACANA CR005996 G013897 337 144 chr4:186234022-186234044GGACAUUCCUUGAAGCAAUG NA CR005997 NA NA 145 chr4:186250330-186250352GUUGGGGUGAUAGGUGCAGA NA CR005999 NA NA 146 chr4:186232147-186232169GAAAACGCCUUCUUCAGAGG NA CR006000 NA NA 147 chr4:186232144-186232166UAUGAAAACGCCUUCUUCAG NA CR006001 NA NA 148 chr4:186250277-186250299CUCAGAUGUGGAUGUUGCCA NA CR006002 NA NA 149 chr4:186254579-186254601CUCUCCUAGGCUUCCCCUGC NA CR006003 NA NA

TABLE 2 Cyno KLKB1 targeted single guide sequences,chromosomal coordinates, and guide sequence homology to human CynoPercent Cyno SEQ ID Exemplary Genomic homology to sgRNA NOCoordinates (mf5) cyno guide sequence human guide G013870 310chr5:185648888-185648908 GCACCUGCUCGACGUACUUU  90 G013871 311chr5:185652966-185652986 AGGAACGCCUACCACUAUAA  90 G013872 312chr5:185688465-185688485 UGAUGGAAACGCUCGGAUGC NA G013873 313chr5:185652964-185652984 AUAGUGGUAGGCGUUCCUCC  90 G013874 314chr5:185652965-185652985 UAUAGUGGUAGGCGUUCCUC  90 G013875 315chr5:185652946-185652966 CUCUUAAAGCACAGUCCCGG  90 G013876 316chr5:185684512-185684532 AAUGCGCCAAACAUCCGGUA 100 G013877 317chr5:185648882-185648902 UUGCCAAAAGUACGUCGAGC  85 G013878 318chr5:185652981-185653001 UAUAAAGGUGCUGAAUAACG  95 G013879 319chr5:185648894-185648914 CGUCGAGCAGGUGCAAUUUC  85 G013880 320chr5:185648883-185648903 UGCUCGACGUACUUUUGGCA  90 G013881 321chr5:185684503-185684523 GGCUUCCCUUACCGGAUGUU  85 G013882 322chr4:186256046-186256066 CAACUGUUGGGUAACUGGAU 100 G013883 323chr5:185652988-185653008 GUGCUGAAUAACGUGGAAUC  95 G013884 324chr5:185680852-185680872 CGGGUAAAUUUUAGAAUGGC 100 G013885 325chr5:185684511-185684531 AUGCGCCAAACAUCCGGUAA 100 G013886 326chr5:185681472-185681492 CUAUGAGUGACCCUCCACAC 100 G013887 327chr5:185679339-185679359 GGCAACAUCCACAUCCGAGA NA G013888 328chr5:185679426-185679446 UUACGUUCUAUACGAAUGCA  85 G013889 329chr5:185652948-185652968 CUCCGGGACUGUGCUUUAAG  90 G013890 330chr5:185681135-185681155 GUCCCAUAUGUAAUCCUAGU  90 G013891 331chr5:185681374-185681394 AAAACAAGCUCACGCAUUGU  95 G013892 332chr5:185652976-185652996 UUCAGCACCUUUAUAGUGGU  90 G013893 333chr5:185681377-185681397 ACAAGCUCACGCAUUGUUGG  95 G013894 334chr5:185679374-185679394 GUUCGACACACAAAAGCAUC  95 G013895 335chr4:186258088-186258108 GGGCGAAGGCUGUGCCCGCA 100 G013896 336chr5:185681481-185681501 GUCACUCAUAGGACACCAGU 100 G013897 337chr5:185647160-185647180 AGCAAGUUCCAUCAAUGACA  95 G013898 338chr5:185679413-185679433 AACGUAAAGAAGAGGCAGCU 100 G013899 339chr5:185651465-185651485 UGCCACCGAGACAUUUAUAA  95 G013900 340chr4:186256017-186256037 GUGUUUGUGUCACCUUUGGA 100 G013901 341chr4:186251792-186251812 GGAUUACAUAUGGGACACAA 100 G013902 342chr5:185681520-185681540 ACACUUACCCAUCAAAGCAG 100 G013903 343chr5:185684660-185684680 CAGUGUAAUUCAAAGGAGCC 100 G013904 344chr5:185681138-185681158 AGGAUUACAUAUGGGACACA 100 G013905 345chr5:185651470-185651490 UUCCUUUAUAAAUGUCUCGG 100 G013906 346chr5:185680632-185680652 CCUCAAGAAAACACCACAUC  95 G013907 347chr5:185688458-185688478 AGAGCAGUGAUGGAAACGCU NA G013908 348chr5:185681129-185681149 UCUCCAACUAGGAUUACAUA  90 G013909 349chr5:185680982-185681002 ACUCCCAGAAGACUGUAAGG NA G013910 350chr5:185679360-185679380 AGCAUCUGGGGCGAGAACUC 100 G013911 351chr5:185679372-185679392 UCGACACACAAAAGCAUCUG NA G013912 352chr5:185681393-185681413 UUGGAGGAACAAACUCUUCU 100 G013913 353chr4:186236797-186236817 UAUAAAGGAAUUGAUAUGAG 100 G013914 354chr4:186256047-186256067 AACUGUUGGGUAACUGGAUG 100 G013915 355chr5:185647095-185647115 AUCUGGCAGUGCUGGGCGUU 100 G013916 356chr4:186258042-186258062 CUUGCAAACACAAUGGAAUG 100 G013917 357chr4:186251628-186251648 UCUCCUCCUUACAGUCUUCU G013918 358chr5:185688296-185688316 CUGUGACUCAGGGAGAUUCA NA G013918 358chr5:185688296-185688316 CUGUGACUCAGGGAGAUUCA 100 G013919 359chr4:186258084-186258104 GGCACAGCCUUCGCCCCAGC 100 G013920 360chr5:185647084-185647104 CUGGGCGUUCGGGGUGUACA 100 G013921 361chr4:186254600-186254620 GAUGUUUGGCGCAUUUAUAG 100 G013922 362chr4:186251771-186251791 CUUCGGAUGGUUCUCCAACU G013923 363chr5:185684517-185684537 CUAUAAAUGCGCCAAACAUC NA G013923 363chr5:185684517-185684537 CUAUAAAUGCGCCAAACAUC 100 G013924 364chr4:186256045-186256065 CCAACUGUUGGGUAACUGGA G013925 365chr5:185680856-185680876 CUCCCGGGUAAAUUUUAGAA 100 G013926 366chr5:185684639-185684659 UAUCGCCUUAAUAAAACUCC  95 G013926 366chr4:186254722-186254742 UAUCGCCUUAAUAAAACUCC 100 G013927 367chr4:186250264-186250284 GCAUCUUGCCUUCUCGGAUG G013928 368chr5:185679421-185679441 UCGUAUAGAACGUAAAGAAG  90 G013929 369chr4:186258038-186258058 CCAUUGUGUUUGCAAGCUAA 100 G013930 370chr5:185651562-185651582 AAAUUGGCAGCGAAUGUUAU  90 G013930 370chr4:186236879-186236899 AAAUUGGCAGCGAAUGUUAU 100 G013931 371chr4:186256048-186256068 CCAUCCAGUUACCCAACAGU 100 G013932 372chr4:186256058-186256078 AACUGGAUGGGGCUUCUCGA 100 G013933 373chr5:185681130-185681150 CUCCAACUAGGAUUACAUAU 100 G013934 374chr5:185679328-185679348 CAUCCGAGAAGGCAAGAUGC  90 G013935 375chr4:186251536-186251556 UUGAAUGUGACUUUCGUUAA 100 G013936 376chr5:185647161-185647181 AUGUCAUUGAUGGAACUUGC  95 G013937 377chr5:185648925-185648945 UUGAUGACCACACUGCUUUA  90 G013938 378chr5:185681470-185681490 CUGUGUGGAGGGUCACUCAU 100 G013939 379chr5:185647112-185647132 GGUGGAAUGUGCACAUCAUC  95 G013940 380chr5:185681105-185681125 UUCUUAAGAUUAUCUUCGGA  90 G013941 381chr5:185685921-185685941 CCUUUGGAAGGUAGGCAUAU 100 G013942 382chr4:186258039-186258059 UCCAUUGUGUUUGCAAGCUA 100 G013943 383chr4:186258008-186258028 CCUGUGACUCAGGGAGAUUC 100 G013944 384chr5:185647103-185647123 UGCACAUCAUCUGGCAGUGC  85 G013945 385chr4:186238299-186238319 GUUAUUCAGCACCUUUAUAG 100 G013946 386chr5:185681480-185681500 GGUCACUCAUAGGACACCAG 100

The guide RNAs identified above as “G0XXXXX” are sgRNAs comprising theidentified 20 nucleotide targeting sequence of Table 1 or Table 2,within the guide structure of SEQ ID NO: 300. In some embodiments, thesgRNA comprises any one of the guide RNAs of Tables 1 or 2 and thenucleotides of SEQ ID NO: 300, optionally wherein the sgRNA comprisesany one of the modification patterns described in Table 4. In someembodiments, the sgRNA comprises any one of the guide RNAs of Tables 1or 2 and any of the conserved portion of sgRNAs of Table 4, optionallywith any one of the modification patterns described in Table 4.

TABLE 3A (Conserved Portion of a spyCas9 sgRNA; SEQ ID NO: 500) 1 2 3 45 6 7 8 9 10 11 12 13 G U U U U A G A G C U A G LS1-LS6 B1-B2 US1-US1214 15 16 17 18 19 20 21 22 23 24 25 26 A A A U A G C A A G U U AUS1-US12 B2-B6 LS7-LS12 27 28 29 30 31 32 33 34 35 36 37 38 39 A A A U AA G G C U A G U LS7-LS12 Nexus 40 41 42 43 44 45 46 47 48 49 50 51 52 CC G U U A U C A A C U U Nexus H1-1 through H1-12 53 54 55 56 57 58 59 6061 62 63 64 65 G A A A A A G U G G C A C H1-1 through H1-12 N H2-1through H2-15 66 67 68 69 70 71 72 73 74 75 76 C G A G U C G G U G CH2-1 through H2-15

TABLE 3B LS1-6 B1-2 US1-12 5′ terminus (n) lower stem n bulge n upperstem B3-6 LS7-12 N1-18 n bulge n lower stem n nexus H1-1 thru H1-12 H2-1thru H2-15 n hairpin 1 n hairpin 2 3′ terminus

In some embodiments, the invention provides a composition comprising oneor more guide RNA (gRNA) comprising guide sequences that direct anRNA-guided DNA binding agent, which can be a nuclease (e.g., a Casnuclease such as Cas9), to a target DNA sequence in KLKB1. The gRNA maycomprise a crRNA comprising a guide sequence shown in Table 1. The gRNAmay comprise a crRNA comprising 17, 18, 19, or 20 contiguous nucleotidesof a guide sequence shown in Table 1. In some embodiments, the gRNAcomprises a crRNA comprising a sequence with about 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% identity to at least 17, 18, 19, or 20contiguous nucleotides of a guide sequence shown in Table 1. In someembodiments, the gRNA comprises a crRNA comprising a sequence with about75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a guidesequence shown in Table 1. The gRNA may further comprise a trRNA. Ineach composition and method embodiment described herein, the crRNA andtrRNA may be associated as a single RNA (sgRNA) or may be on separateRNAs (dgRNA). In the context of sgRNAs, the crRNA and trRNA componentsmay be covalently linked, e.g., via a phosphodiester bond or othercovalent bond.

In each of the compositions, use, and method embodiments describedherein, the guide RNA may comprise two RNA molecules as a “dual guideRNA” or “dgRNA.” The dgRNA comprises a first RNA molecule comprising acrRNA comprising, e.g., a guide sequence shown in Table 1, and a secondRNA molecule comprising a trRNA. The first and second RNA molecules maynot be covalently linked, but may form an RNA duplex via the basepairing between portions of the crRNA and the trRNA.

In each of the composition, use, and method embodiments describedherein, the guide RNA may comprise a single RNA molecule as a “singleguide RNA” or “sgRNA”. The sgRNA may comprise a crRNA (or a portionthereof) comprising a guide sequence shown in Table 1 covalently linkedto a trRNA. The sgRNA may comprise 17, 18, 19, or 20 contiguousnucleotides of a guide sequence shown in Table 1. In some embodiments,the crRNA and the trRNA are covalently linked via a linker. In someembodiments, the sgRNA forms a stem-loop structure via the base pairingbetween portions of the crRNA and the trRNA. In some embodiments, thecrRNA and the trRNA are covalently linked via one or more bonds that arenot a phosphodiester bond.

In some embodiments, the trRNA may comprise all or a portion of a trRNAsequence derived from a naturally-occurring CRISPR/Cas system. In someembodiments, the trRNA comprises a truncated or modified wild typetrRNA. The length of the trRNA depends on the CRISPR/Cas system used. Insome embodiments, the trRNA comprises or consists of 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90,100, or more than 100 nucleotides. In some embodiments, the trRNA maycomprise certain secondary structures, such as, for example, one or morehairpin or stem-loop structures, or one or more bulge structures.

In some embodiments, a composition comprising one or more guide RNAscomprising a guide sequence of any one of SEQ ID NOs: 1-149 is provided.In some embodiments, a composition comprising one or more guide RNAscomprising a guide sequence of any one of SEQ ID Nos: 1-149 and anyconserved portion of an sgRNA shown in Table 4, optionally having amodification pattern of any of an sgRNA shown in Table 4, optionallywherein the sgRNA comprises a 5′ and 3′ end modification (if not alreadyshown in the construct of Table 4) is provided. In some embodiments, acomposition comprising one or more guide RNAs comprising a guidesequence of any one of SEQ ID Nos: 1-149 is provided, wherein thenucleotides of SEQ ID NO: 170, 171, 172, or 173 follow the guidesequence at its 3′ end. In some embodiments, the one or more guide RNAscomprising a guide sequence of any one of SEQ ID Nos: 1-149, wherein thenucleotides of SEQ ID NO: 170, 171, 172, or 173 follow the guidesequence at its 3′ end, is modified according to the modificationpattern of SEQ ID NO: 300.

In some embodiments, a composition comprising one or more guide RNAscomprising a guide sequence of any one of SEQ ID NOs: 1-149 is provided.In one aspect, a composition comprising one or more gRNAs is provided,comprising a guide sequence that is at least 99%, 98%, 97%, 96%, 95%,94%, 93%, 92%, 91%, or 90% identical to any of the nucleic acids of SEQID NOs: 1-149.

In other embodiments, a composition is provided that comprises at leastone, e.g., at least two gRNA's comprising guide sequences selected fromany two or more of the guide sequences of SEQ ID NOs: 1-149. In someembodiments, the composition comprises at least two gRNA's that eachcomprise a guide sequence at least 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, or 90% identical to any of the nucleic acids of SEQ ID NOs:1-149.

The guide RNA compositions of the present invention are designed torecognize (e.g., hybridize to) a target sequence in the KLKB1 gene. Forexample, the KLKB1 target sequence may be recognized and cleaved by aprovided Cas cleavase comprising a guide RNA. In some embodiments, anRNA-guided DNA binding agent, such as a Cas cleavase, may be directed bya guide RNA to a target sequence of the KLKB1 gene, where the guidesequence of the guide RNA hybridizes with the target sequence and theRNA-guided DNA binding agent, such as a Cas cleavase, cleaves the targetsequence.

In some embodiments, the selection of the one or more guide RNAs isdetermined based on target sequences within the KLKB1 gene. In someembodiments, the compositions comprising one or more guide sequencescomprise a guide sequence that is complementary to the correspondinggenomic region shown in Table 1 below, according to coordinates fromhuman reference genome hg38. Guide sequences of further embodiments maybe complementary to sequences in the close vicinity of the genomiccoordinate listed in any of the Tables provided herein. For example,guide sequences of further embodiments may be complementary to sequencesthat comprise 15 consecutive nucleotides f10 nucleotides of a genomiccoordinate listed in any of the Tables disclosed herein.

Without being bound by any particular theory, mutations (e.g.,frameshift mutations resulting from indels occurring as a result of anuclease-mediated DSB) in certain regions of the gene may be lesstolerable than mutations in other regions of the gene, thus the locationof a DSB is an important factor in the amount or type of proteinknockdown that may result. In some embodiments, a gRNA complementary orhaving complementarity to a target sequence within KLKB1 is used todirect the RNA-guided DNA binding agent to a particular location in theKLKB1 gene. In some embodiments, gRNAs are designed to have guidesequences that are complementary or have complementarity to targetsequences in exon 1, exon 3, exon 4, exon 5, exon 6, exon 8, exon 9,exon 10, exon 11, exon 12, exon 13, exon 14, or exon 15 of KLKB1.

In some embodiments, the guide sequence is at least 99%, 98%, 97%, 96%,95%, 94%, 93%, 92%, 91%, or 90% identical to a target sequence presentin the human KLKB1 gene. In some embodiments, the target sequence may becomplementary to the guide sequence of the guide RNA. In someembodiments, the degree of complementarity or identity between a guidesequence of a guide RNA and its corresponding target sequence may be atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In someembodiments, the target sequence and the guide sequence of the gRNA maybe 100% complementary or identical. In other embodiments, the targetsequence and the guide sequence of the gRNA may contain at least onemismatch. For example, the target sequence and the guide sequence of thegRNA may contain 1, 2, 3, or 4 mismatches, where the total length of theguide sequence is 20. In some embodiments, the target sequence and theguide sequence of the gRNA may contain 1-4 mismatches where the guidesequence is 20 nucleotides.

In some embodiments, a composition or formulation disclosed hereincomprises an mRNA comprising an open reading frame (ORF) encoding anRNA-guided DNA binding agent, such as a Cas nuclease as describedherein. In some embodiments, an mRNA comprising an ORF encoding anRNA-guided DNA binding agent, such as a Cas nuclease, is provided, used,or administered.

B. Modified gRNAs and mRNAs

In some embodiments, the gRNA is chemically modified. A gRNA comprisingone or more modified nucleosides or nucleotides is called a “modified”gRNA or “chemically modified” gRNA, to describe the presence of one ormore non-naturally and/or naturally occurring components orconfigurations that are used instead of or in addition to the canonicalA, G, C, and U residues. In some embodiments, a modified gRNA issynthesized with a non-canonical nucleoside or nucleotide, is herecalled “modified.” Modified nucleosides and nucleotides can include oneor more of: (i) alteration, e.g., replacement, of one or both of thenon-linking phosphate oxygens and/or of one or more of the linkingphosphate oxygens in the phosphodiester backbone linkage (an exemplarybackbone modification); (ii) alteration, e.g., replacement, of aconstituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribosesugar (an exemplary sugar modification); (iii) wholesale replacement ofthe phosphate moiety with “dephospho” linkers (an exemplary backbonemodification); (iv) modification or replacement of a naturally occurringnucleobase, including with a non-canonical nucleobase (an exemplary basemodification); (v) replacement or modification of the ribose-phosphatebackbone (an exemplary backbone modification); (vi) modification of the3′ end or 5′ end of the oligonucleotide, e.g., removal, modification orreplacement of a terminal phosphate group or conjugation of a moiety,cap or linker (such 3′ or 5′ cap modifications may comprise a sugarand/or backbone modification); and (vii) modification or replacement ofthe sugar (an exemplary sugar modification).

Chemical modifications such as those listed above can be combined toprovide modified gRNAs and/or mRNAs comprising nucleosides andnucleotides (collectively “residues”) that can have two, three, four, ormore modifications. For example, a modified residue can have a modifiedsugar and a modified nucleobase. In some embodiments, every base of agRNA is modified, e.g., all bases have a modified phosphate group, suchas a phosphorothioate group. In certain embodiments, all, orsubstantially all, of the phosphate groups of an gRNA molecule arereplaced with phosphorothioate groups. In some embodiments, modifiedgRNAs comprise at least one modified residue at or near the 5′ end ofthe RNA. In some embodiments, modified gRNAs comprise at least onemodified residue at or near the 3′ end of the RNA.

In some embodiments, the gRNA comprises one, two, three or more modifiedresidues. In some embodiments, at least 5% (e.g., at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or 100%) of the positions in a modifiedgRNA are modified nucleosides or nucleotides.

Unmodified nucleic acids can be prone to degradation by, e.g.,intracellular nucleases or those found in serum. For example, nucleasescan hydrolyze nucleic acid phosphodiester bonds. Accordingly, in oneaspect the gRNAs described herein can contain one or more modifiednucleosides or nucleotides, e.g., to introduce stability towardintracellular or serum-based nucleases. In some embodiments, themodified gRNA molecules described herein can exhibit a reduced innateimmune response when introduced into a population of cells, both in vivoand ex vivo. The term “innate immune response” includes a cellularresponse to exogenous nucleic acids, including single stranded nucleicacids, which involves the induction of cytokine expression and release,particularly the interferons, and cell death.

In some embodiments of a backbone modification, the phosphate group of amodified residue can be modified by replacing one or more of the oxygenswith a different substituent. Further, the modified residue, e.g.,modified residue present in a modified nucleic acid, can include thewholesale replacement of an unmodified phosphate moiety with a modifiedphosphate group as described herein. In some embodiments, the backbonemodification of the phosphate backbone can include alterations thatresult in either an uncharged linker or a charged linker withunsymmetrical charge distribution.

Examples of modified phosphate groups include, phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. The phosphorous atom in an unmodified phosphate groupis achiral. However, replacement of one of the non-bridging oxygens withone of the above atoms or groups of atoms can render the phosphorousatom chiral. The stereogenic phosphorous atom can possess either the “R”configuration (herein Rp) or the “S” configuration (herein Sp). Thebackbone can also be modified by replacement of a bridging oxygen,(i.e., the oxygen that links the phosphate to the nucleoside), withnitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates)and carbon (bridged methylenephosphonates). The replacement can occur ateither linking oxygen or at both of the linking oxygens.

The phosphate group can be replaced by non-phosphorus containingconnectors in certain backbone modifications. In some embodiments, thecharged phosphate group can be replaced by a neutral moiety. Examples ofmoieties which can replace the phosphate group can include, withoutlimitation, e.g., methyl phosphonate, hydroxylamino, siloxane,carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxidelinker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime,methyleneimino, methylenemethylimino, methylenehydrazo,methylenedimethylhydrazo and methyleneoxymethylimino.

Scaffolds that can mimic nucleic acids can also be constructed whereinthe phosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates. Such modifications may comprisebackbone and sugar modifications. In some embodiments, the nucleobasescan be tethered by a surrogate backbone. Examples can include, withoutlimitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleicacid (PNA) nucleoside surrogates.

The modified nucleosides and modified nucleotides can include one ormore modifications to the sugar group, i.e. a sugar modification. Forexample, the 2′ hydroxyl group (OH) can be modified, e.g. replaced witha number of different “oxy” or “deoxy” substituents. In someembodiments, modifications to the 2′ hydroxyl group can enhance thestability of the nucleic acid since the hydroxyl can no longer bedeprotonated to form a 2′-alkoxide ion.

Examples of 2′ hydroxyl group modifications can include alkoxy oraryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl,heteroaryl or a sugar); polyethyleneglycols (PEG),O(CH₂CH₂O)_(n)CH₂CH₂OR wherein R can be, e.g., H or optionallysubstituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8,from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4to 16, and from 4 to 20). In some embodiments, the 2′ hydroxyl groupmodification can be 2′-O-Me. In some embodiments, the 2′ hydroxyl groupmodification can be a 2′-fluoro modification, which replaces the 2′hydroxyl group with a fluoride. In some embodiments, the 2′ hydroxylgroup modification can include “locked” nucleic acids (LNA) in which the2′ hydroxyl can be connected, e.g., by a C₁₋₆ alkylene or C₁₋₆heteroalkylene bridge, to the 4′ carbon of the same ribose sugar, whereexemplary bridges can include methylene, propylene, ether, or aminobridges; O-amino (wherein amino can be, e.g., NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, ordiheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy,O(CH₂)_(n)-amino, (wherein amino can be, e.g., NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, ordiheteroarylamino, ethylenediamine, or polyamino). In some embodiments,the 2′ hydroxyl group modification can include “unlocked” nucleic acids(UNA) in which the ribose ring lacks the C2′-C3′ bond. In someembodiments, the 2′ hydroxyl group modification can include themethoxyethyl group (MOE), (OCH₂CH₂OCH₃, e.g., a PEG derivative).

“Deoxy” 2′ modifications can include hydrogen (i.e. deoxyribose sugars,e.g., at the overhang portions of partially dsRNA); halo (e.g., bromo,chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino,heteroarylamino, diheteroarylamino, or amino acid);NH(CH₂CH₂NH)_(n)CH2CH₂- amino (wherein amino can be, e.g., as describedherein), —NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl,aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino as described herein.

The sugar modification can comprise a sugar group which may also containone or more carbons that possess the opposite stereochemicalconfiguration than that of the corresponding carbon in ribose. Thus, amodified nucleic acid can include nucleotides containing e.g.,arabinose, as the sugar. The modified nucleic acids can also includeabasic sugars. These abasic sugars can also be further modified at oneor more of the constituent sugar atoms. The modified nucleic acids canalso include one or more sugars that are in the L form, e.g.L-nucleosides.

The modified nucleosides and modified nucleotides described herein,which can be incorporated into a modified nucleic acid, can include amodified base, also called a nucleobase. Examples of nucleobasesinclude, but are not limited to, adenine (A), guanine (G), cytosine (C),and uracil (U). These nucleobases can be modified or wholly replaced toprovide modified residues that can be incorporated into modified nucleicacids. The nucleobase of the nucleotide can be independently selectedfrom a purine, a pyrimidine, a purine analog, or pyrimidine analog. Insome embodiments, the nucleobase can include, for example,naturally-occurring and synthetic derivatives of a base.

In embodiments employing a dual guide RNA, each of the crRNA and thetracr RNA can contain modifications. Such modifications may be at one orboth ends of the crRNA and/or tracr RNA. In embodiments comprising ansgRNA, one or more residues at one or both ends of the sgRNA may bechemically modified, and/or internal nucleosides may be modified, and/orthe entire sgRNA may be chemically modified. Certain embodimentscomprise a 5′ end modification. Certain embodiments comprise a 3′ endmodification.

In some embodiments, the guide RNAs disclosed herein comprise one of themodification patterns disclosed in WO2018/107028 and/or WO2019/237069,the contents of which are hereby incorporated by reference in theirentirety. For example, the guide RNAs disclosed herein may comprise theshort-guide structure described at claims 1-15 and/or the modificationpatterns described at claims 16-462 of WO2019/237069. In someembodiments, the guide RNAs disclosed herein comprise one of thestructures/modification patterns disclosed in WO 2015/200555, thecontents of which are hereby incorporated by reference in theirentirety. In some embodiments, the guide RNAs disclosed herein compriseone of the structures/modification patterns disclosed in WO2017/136794,the contents of which are hereby incorporated by reference in theirentirety.

C. YA Modifications

A modification at a YA site (also referred to herein as “YAmodification”) can be a modification of the internucleoside linkage, amodification of the base (pyrimidine or adenine), e.g. by chemicalmodification, substitution, or otherwise, and/or a modification of thesugar (e.g. at the 2′ position, such as 2′-O-alkyl, 2′-F, 2′-moe, 2′-Farabinose, 2′-H (deoxyribose), and the like). In some embodiments, a “YAmodification” is any modification that alters the structure of thedinucleotide motif to reduce RNA endonuclease activity, e.g., byinterfering with recognition or cleavage of a YA site by an RNase and/orby stabilizing an RNA structure (e.g., secondary structure) thatdecreases accessibility of a cleavage site to an RNase. See Peacock etal., J Org Chem. 76: 7295-7300 (2011); Behlke, Oligonucleotides18:305-320 (2008); Ku et al., Adv. Drug Delivery Reviews 104: 16-28(2016); Ghidini et al., Chem. Commun., 2013, 49, 9036. Peacock et al.,Belhke, Ku, and Ghidini provide exemplary modifications suitable as YAmodifications. Modifications known to those of skill in the art toreduce endonucleolytic degradation are encompassed. Exemplary 2′ ribosemodifications that affect the 2′ hydroxyl group involved in RNasecleavage are 2′-H and 2′-O-alkyl, including 2′-O-Me. Modifications suchas bicyclic ribose analogs, UNA, and modified internucleoside linkagesof the residues at the YA site can be YA modifications. Exemplary basemodifications that can stabilize RNA structures are pseudouridine and5-methylcytosine. In some embodiments, at least one nucleotide of the YAsite is modified. In some embodiments, the pyrimidine (also called“pyrimidine position”) of the YA site comprises a modification (whichincludes a modification altering the internucleoside linkage immediately3′ of the sugar of the pyrimidine, a modification of the pyrimidinebase, and a modification of the ribose, e.g. at its 2′ position). Insome embodiments, the adenine (also called “adenine position”) of the YAsite comprises a modification (which includes a modification alteringthe internucleoside linkage immediately 3′ of the sugar of thepyrimidine, a modification of the pyrimidine base, and a modification ofthe ribose, e.g. at its 2′ position). In some embodiments, thepyrimidine and the adenine of the YA site comprise modifications. Insome embodiments, the YA modification reduces RNA endonuclease activity.

In some embodiments, an sgRNA comprises modifications at 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more YA sites. In someembodiments, the pyrimidine of the YA site comprises a modification(which includes a modification altering the internucleoside linkageimmediately 3′ of the sugar of the pyrimidine). In some embodiments, theadenine of the YA site comprises a modification (which includes amodification altering the internucleoside linkage immediately 3′ of thesugar of the adenine). In some embodiments, the pyrimidine and theadenine of the YA site comprise modifications, such as sugar, base, orinternucleoside linkage modifications. The YA modifications can be anyof the types of modifications set forth herein. In some embodiments, theYA modifications comprise one or more of phosphorothioate, 2′-OMe, or2′-fluoro. In some embodiments, the YA modifications comprise pyrimidinemodifications comprising one or more of phosphorothioate, 2′-OMe, or2′-fluoro. In some embodiments, the YA modification comprises a bicyclicribose analog (e.g., an LNA, BNA, or ENA) within an RNA duplex regionthat contains one or more YA sites. In some embodiments, the YAmodification comprises a bicyclic ribose analog (e.g., an LNA, BNA, orENA) within an RNA duplex region that contains a YA site, wherein the YAmodification is distal to the YA site.

In some embodiments, the sgRNA comprises a guide region YA sitemodification. In some embodiments, the guide region comprises 1, 2, 3,4, 5, or more YA sites (“guide region YA sites”) that may comprise YAmodifications. In some embodiments, one or more YA sites located at5-end, 6-end, 7-end, 8-end, 9-end, or 10-end from the 5′ end of the 5′terminus (where “5-end”, etc., refers to position 5 to the 3′ end of theguide region, i.e., the most 3′ nucleotide in the guide region) compriseYA modifications. In some embodiments, two or more YA sites located at5-end, 6-end, 7-end, 8-end, 9-end, or 10-end from the 5′ end of the 5′terminus comprise YA modifications. In some embodiments, three or moreYA sites located at 5-end, 6-end, 7-end, 8-end, 9-end, or 10-end fromthe 5′ end of the 5′ terminus comprise YA modifications. In someembodiments, four or more YA sites located at 5-end, 6-end, 7-end,8-end, 9-end, or 10-end from the 5′ end of the 5′ terminus comprise YAmodifications. In some embodiments, five or more YA sites located at5-end, 6-end, 7-end, 8-end, 9-end, or 10-end from the 5′ end of the 5′terminus comprise YA modifications. A modified guide region YA sitecomprises a YA modification.

In some embodiments, a modified guide region YA site is within 17, 16,15, 14, 13, 12, 11, 10, or 9 nucleotides of the 3′ terminal nucleotideof the guide region. For example, if a modified guide region YA site iswithin 10 nucleotides of the 3′ terminal nucleotide of the guide regionand the guide region is 20 nucleotides long, then the modifiednucleotide of the modified guide region YA site is located at any ofpositions 11-20. In some embodiments, a YA modification is locatedwithin a YA site 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 nucleotides from the 3′ terminal nucleotide of theguide region. In some embodiments, a YA modification is located 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1nucleotides from the 3′ terminal nucleotide of the guide region.

In some embodiments, a modified guide region YA site is at or afternucleotide 4, 5, 6, 7, 8, 9, 10, or 11 from the 5′ end of the 5′terminus.

In some embodiments, a modified guide region YA site is other than a 5′end modification. For example, an sgRNA can comprise a 5′ endmodification as described herein and further comprise a modified guideregion YA site. Alternatively, an sgRNA can comprise an unmodified 5′end and a modified guide region YA site. Alternatively, an sgRNA cancomprise a modified 5′ end and an unmodified guide region YA site.

In some embodiments, a modified guide region YA site comprises amodification that at least one nucleotide located 5′ of the guide regionYA site does not comprise. For example, if nucleotides 1-3 comprisephosphorothioates, nucleotide 4 comprises only a 2′-OMe modification,and nucleotide 5 is the pyrimidine of a YA site and comprises aphosphorothioate, then the modified guide region YA site comprises amodification (phosphorothioate) that at least one nucleotide located 5′of the guide region YA site (nucleotide 4) does not comprise. In anotherexample, if nucleotides 1-3 comprise phosphorothioates, and nucleotide 4is the pyrimidine of a YA site and comprises a 2′-OMe, then the modifiedguide region YA site comprises a modification (2′-OMe) that at least onenucleotide located 5′ of the guide region YA site (any of nucleotides1-3) does not comprise. This condition is also always satisfied if anunmodified nucleotide is located 5′ of the modified guide region YAsite.

In some embodiments, the modified guide region YA sites comprisemodifications as described for YA sites above.

Additional embodiments of guide region YA site modifications are setforth in the summary above. Any embodiments set forth elsewhere in thisdisclosure may be combined to the extent feasible with any of theforegoing embodiments.

In some embodiments, the sgRNA comprises a conserved region YA sitemodification. Conserved region YA sites 1-10 are illustrated in FIG. 14. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved regionYA sites comprise modifications.

In some embodiments, conserved region YA sites 1, 8, or 1 and 8 compriseYA modifications. In some embodiments, conserved region YA sites 1, 2,3, 4, and 10 comprise YA modifications. In some embodiments, YA sites 2,3, 4, 8, and 10 comprise YA modifications. In some embodiments,conserved region YA sites 1, 2, 3, and 10 comprise YA modifications. Insome embodiments, YA sites 2, 3, 8, and 10 comprise YA modifications. Insome embodiments, YA sites 1, 2, 3, 4, 8, and 10 comprise YAmodifications. In some embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 additionalconserved region YA sites comprise YA modifications.

In some embodiments, 1, 2, 3, or 4 of conserved region YA sites 2, 3, 4,and 10 comprise YA modifications. In some embodiments, 1, 2, 3, 4, 5, 6,7, or 8 additional conserved region YA sites comprise YA modifications.

In some embodiments, the modified conserved region YA sites comprisemodifications as described for YA sites above.

Additional embodiments of conserved region YA site modifications are setforth in the summary above. Any embodiments set forth elsewhere in thisdisclosure may be combined to the extent feasible with any of theforegoing embodiments.

In some embodiments, an sgRNA comprising the guide sequence of any oneof SEQ ID NOs: 1-149 and any conserved portion of an sgRNA shown inTable 4, optionally having a modification pattern of any of an sgRNAshown in Table 4, optionally wherein the sgRNA comprises a 5′ and 3′ endmodification (if not already shown in the construct of Table 4) isprovided.

In some embodiments, the sgRNA comprises any of the modificationpatterns shown below in Table 4, where N is any natural or non-naturalnucleotide, and wherein the totality of the N's comprise a KLKB1 guidesequence as described herein in Table 1. Table 4 does not depict theguide sequence portion of the sgRNA. The modifications remain as shownin Table 4 despite the substitution of N's for the nucleotides of aguide. That is, although the nucleotides of the guide replace the “N's”,the nucleotides are modified as shown in Table 4.

TABLE 4 sgRNA modification patterns and conserved portionsof an sgRNA. The guide sequence is not shown and willappend the shown sequence at its 5′ end. SEQ ID NO Name Sequence 171Exemplary GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC conservedAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU portion 172 ExemplaryGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC conservedAACUUGAAAAAGUGGCACCGAGUCGGUGC portion 173 ExemplaryGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC conservedAACUUGGCACCGAGUCGGUGC portion 170 ExemplaryGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC conservedAACUUGGCACCGAGUCGGUGCUUUU portion 168 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGAAAAAGUGGCACCGAGUCGGUGCmU*mU*mU*U 169 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGGUGCmU*mU*mU*U 400 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGAAAAAGUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCm U*mU*mU*mU 401 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmU mCmGmGmUmGmCmU*mU*mU*mU402 Exemplary- GUUUUAGAGCUAmGmAmAmAUAGCAAGUUAAAAUAAGGCUAGUCCGU mod onlyUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU*mU*mU*U 403 Exemplary-GUUUUAGAmGmCmUmAGAAAmUmAmGmCAAGUUAAAAUAAGGCUAG mod onlyUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU*mU*mU*U 404 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU*mU* mU*U 405 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 406 Exemplary-mGUUUUmAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAA mod onlyGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 407 Exemplary-fGfUfUfUfUfAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUA mod onlyAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmU*mU*mU 408 Exemplary-mGfUfUfUfUmAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAU mod onlyAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 409 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAAAmAmU mod onlyAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 410 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAfAfAmAm mod onlyUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmU*mU*mU 411 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUfUmAfAmAfAm mod onlyUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 412 ExemplarymGUUUUmAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAAAmA mod onlymUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 413 Exemplary-mGUUUUmAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAfAfAm mod onlyAmUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 414 Exemplary-mGUUUUmAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUfUmAfAmAf mod onlyAmUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 415 Exemplary-fGfUfUfUfUfAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAAAm mod onlyAmUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 416 Exemplary-fGfUfUfUfUfAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAfAfA mod onlymAmUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 417 Exemplary-fGfUfUfUfUfAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUfUmAfAmAf mod onlyAmUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 418 Exemplary-GUUUUAmGmAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAA mod onlyGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 419 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCmAmAmGmUUAAAAU mod onlyAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 420 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUfAfUfCfAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 421 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAmUmCmAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 422 Exemplary-fGfUfUfUfUfAmGmAmGmCmUmAmGmAmAmAmUmAmGmCmAmAmGmUm mod onlyUmAfAfAmAmUAAGGCUAGUCCGUUAmUmCmAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU* mU*mU 423 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU 424 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmU*mU*mU 425 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGfUfCfGfGfUfGfCfU*fU*fU*mU 426 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCAfAmCfUmUfGmAfAmAfAmAfGmUfGmGfCmAfCmCfGmAfGmUfCmGfGmUfGmCfU*mU*fU*mU 427 Exemplary-mGUUUUmAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAA mod onlyGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 428 ExemplaryfGfUfUfUfUfAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUA mod onlyAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 429 Exemplary-mGfUfUfUfUmAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAU mod onlyAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 430 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAAAmAmU mod onlyAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 431 ExemplaryGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAfAfAmAm mod onlyUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 432 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUfUmAfAmAfAm mod onlyUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 433 ExemplarymGUUUUmAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAAAmA mod onlymUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 434 Exemplary-mGUUUUmAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAfAfAm mod onlyAmUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 435 Exemplary-mGUUUUmAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUfUmAfAmAf mod onlyAmUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 436 Exemplary-fGfUfUfUfUfAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAAAm mod onlyAmUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 437 Exemplary-fGfUfUfUfUfAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAfAfA mod onlymAmUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 438 Exemplary-fGfUfUfUfUfAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUfUmAfAmAf mod onlyAmUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 439 Exemplary-GUUUUAmGmAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAA mod onlyGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 440 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCmAmAmGmUUAAAAU mod onlyAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 441 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUfAfUfCfAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 442 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAmUmCmAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 443 Exemplary-fGfUfUfUfUfAmGmAmGmCmUmAmGmAmAmAmUmAmGmCmAmAmGmUm mod onlyUmAfAfAmAmUAAGGCUAGUCCGUUAmUmCmAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU* mU*mU 444 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU 445 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmU*mU*mU 446 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGfUfCfGfGfUfGfCfU*fU*fU*mU 447 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCAfAmCfUmUfGmAfAmAfAmAfGmUfGmGfCmAfCmCfGmAfGmUfCmGfGmUfGmCfU*mU*fU*mU 448 Exemplary-mN*mN*mN*mNNN*N*fN*fN*fN*fNNfNfNNNfNfNNN guide region mod only 449Exemplary- mN*mN*mN*mNNN*N*fN*fN*fN*fNNfNfNNN*fNfNNN guide regionmod only 450 Exemplary- GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGmod only CUAGUCCGUUAUCAACUUGGCACCGAGUCGG*mU*mG*mC 174 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUAAGCACCGAGUCGG*mU*mG*mC 175 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUCAGCACCGAGUCGG*mU*mG*mC 176 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyCACUUGGCACCGAGUCGG*mU*mG*mC 177 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUACGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 178 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAAGAGCUGGCACCGAGUCGG*mU*mG*mC 179 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAAGAAAUGGCACCGAGUCGG*mU*mG*mC 180 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyACGAAAGGGCACCGAGUCGG*mU*mG*mC 181 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAAAAAUGGCACCGAGUCGG*mU*mG*mC 182 ExemplaryGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAAAAGUGGCACCGAGUCGG*mU*mG*mC 183 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACAGUGGCACCGAGUCGG*mU*mG*mC 184 ExemplaryGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyACAAGGGCACCGAGUCGG*mU*mG*mC 185 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAAAAUGGCACCGAGUCGG*mU*mG*mC 186 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAAAGGCACCGAGUCGG*mU*mG*mC 187 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAAGGGCACCGAGUCGG*mU*mG*mC 188 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAGGCACCGAGUCGG*mU*mG*mC 189 Exemplary-GUUUUAGAGCUAGAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA mod onlyACUUGGCACCGAGUCGG*mU*mG*mC 190 ExemplaryGUUUUAGAGCGCAAAGCGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA mod onlyACUUGGCACCGAGUCGG*mU*mG*mC 191 Exemplary-GUUUUAGAGCGCGAAGCGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA mod onlyACUUGGCACCGAGUCGG*mU*mG*mC 192 ExemplaryGUUUUAGAGCGGAAACGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA mod onlyCUUGGCACCGAGUCGGU*mG*mC*mU 193 Exemplary-GUUUUAGAGCGGAAACGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA mod onlyCUUGGCACCGAGUCGG*mU*mG*mC 194 Exemplary-GUUUUAGAGCCGAAAGGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA mod onlyCUUGGCACCGAGUCGG*mU*mG*mC 195 Exemplary-GUUUUAGAGCUGAAAAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA mod onlyCUUGGCACCGAGUCGG*mU*mG*mC 196 Exemplary-GUUUUAGAGCGAAAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU mod onlyUGGCACCGAGUCGG*mU*mG*mC 197 Exemplary-GUUUUAGAGCGAAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU mod onlyGGCACCGAGUCGG*mU*mG*mC 198 Exemplary-GUUUUAGAGCAAAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU mod onlyGGCACCGAGUCGG*mU*mG*mC 199 Exemplary-GUUUUAGAGGAAACAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG mod onlyGCACCGAGUCGG*mU*mG*mC 202 Exemplary-GUUUUAGAGCAAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG mod onlyGCACCGAGUCGG*mU*mG*mC 203 Exemplary-GUUUUAGAGCGAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG mod onlyGCACCGAGUCGG*mU*mG*mC 204 Exemplary-GUUUUAGAGCGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGC mod onlyACCGAGUCGG*mU*mG*mC 205 Exemplary-GUUUUAGAGAACAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGC mod onlyACCGAGUCGG*mU*mG*mC 206 Exemplary-GUUUUAGAGACAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGCA mod onlyCCGAGUCGG*mU*mG*mC 207 Exemplary-GUUUUAGAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGCAC mod onlyCGAGUCGG*mU*mG*mC 208 Exemplary-GUUUUAGAAAAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGCAC mod onlyCGAGUCGG*mU*mG*mC 209 Exemplary-GUUUUAGAAAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGCACC mod onlyGAGUCGG*mU*mG*mC 210 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUCG*mG*mU*mG 211 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUC*mG*mG*mU 212 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGU*mC*mG*mG 213 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAG*mU*mC*mG 214 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGA*mG*mU*mC 215 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCG*mA*mG*mU 216 Exemplary-GUUUUAGAGCGAAAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCACUG mod onlyGCACCGAGUCGG*mU*mG*mC 217 Exemplary-GUUUUAGAGCGAAAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAGGC mod onlyACCGAGUCGG*mU*mG*mC 218 Exemplary-GUUUUAGAGAAAAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGC mod onlyACCGAGUCGG*mU*mG*mC 219 Exemplary-GUUUUAGAGGAAACAAGUUAAAAUAAGGCUAGUCCGUUAUCAAUGGC mod onlyACCGAGUCGG*mU*mG*mC 220 Exemplary-GUUUUAGAGAAAAAGUUAAAAUAAGGCUAGUCCGUUAUCAAUGGCAC mod onlyCGAGUCGG*mU*mG*mC 221 Exemplary-GUUUUAGAGGAAACAAGUUAAAAUAAGGCUAGUCCGUUAUCACUGGC mod onlyACCGAGUCGG*mU*mG*mC 222 Exemplary-GUUUUAGAGAAAAAGUUAAAAUAAGGCUAGUCCGUUAUCAGGCACCG mod only AGUCGG*mU*mG*mC223 Exemplary- GUUUUAGAGCGAAAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAGC mod onlyUAUGGCACCGAGUCGG*mU*mG*mC 224 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCAGUCCGUUAUCA mod onlyACUUGGCACCGAGUCGG*mU*mG*mC 225 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUGUCCGUUAUCA mod onlyACUUGGCACCGAGUCGG*mU*mG*mC 226 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCGUCCGUUAUCAA mod onlyCUUGGCACCGAGUCGG*mU*mG*mC 227 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGUAUCCGUUAUCAA mod onlyCUUGGCACCGAGUCGG*mU*mG*mC 228 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGUUCCGUUAUCAAC mod onlyUUGGCACCGAGUCGG*mU*mG*mC 229 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGAUCCGUUAUCAAC mod onlyUUGGCACCGAGUCGG*mU*mG*mC 230 Exemplary-GUUUUCGAGCUAGAAAUAGCAAGUGAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 231 Exemplary-GUUUUUGAGCUAGAAAUAGCAAGUAAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 232 Exemplary-GUUUUAGAGCGAGAAAUCGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 233 Exemplary-GUUUUAGAGCUAGAAAUAGCGAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 234 Exemplary-GUUUUAGAGCUAGAAAUAGCCGGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 235 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUGAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 236 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUGGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 237 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUCGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 238 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUUGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 239 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUGUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 240 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUCUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 241 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUUUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 242 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUG mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 243 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGGACCGAGUCGG*mU*mC*mC 244 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGAACCGAGUCGG*mU*mU*mC 245 ExemplaryGUUUUCGAGCGAGAAAUCGCGAGUGAAAAUGAGGCUGGUCCGUUGUG mod onlyAACUUGGAACCGAGUCGG*mU*mU*mC 246 Exemplary-GUUUUUGAGCGAGAAAUCGCAAGUAAAAAUAAGGCUCGUCCGUUCUG mod onlyAACUUGGAACCGAGUCGG*mU*mU*mC 247 Exemplary-GUUUCGGAGCCGGAAACGGCGAGUCGAAAUGAGGCUGGUCCGUUGUCG mod onlyGCUCGGAACCGAGUCGG*mU*mU*mC 248 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 249 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 250 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 251 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 252 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 253 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 254 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 255 Exemplary-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC mod onlyAACUUGGCACCGAGUCGG*mU*mG*mC 256 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCACGAAAGGGCACCGAGUCGG*mU*mG*mC 257 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCAAAAAUGGCACCGAGUCGG*mU*mG*mC 258 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCACAAGGGCACCGAGUCGG*mU*mG*mC 259 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCAAAAUGGCACCGAGUCGG*mU*mG*mC 260 Exemplary-GUUUUAGAGCGCGAAGCGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA mod onlyAAAUGGCACCGAGUCGG*mU*mG*mC 261 Exemplary-GUUUUAGAGCUGAAAAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA mod onlyAAUGGCACCGAGUCGG*mU*mG*mC 262 Exemplary-GUUUUAGAGCGAAAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAAA mod onlyUGGCACCGAGUCGG*mU*mG*mC 263 Exemplary-GUUUUAGAGCAAAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAAAU mod onlyGGCACCGAGUCGG*mU*mG*mC 264 Exemplary-GUUUUAGAmGmCmGmAmAmAmGmCAAGUUAAAAUAAGGCUAGUCCGU mod onlyUAUCAACUUGGCACCGAGUCGG*mU*mG*mC 265 Exemplary-GUUUUAGAmGmCmGmAmAmAmGmCAAGUUAAAAUAAGGCUAGUCCGU mod onlyUAUCAAGAAAUGGCACCGAGUCGG*mU*mG*mC 266 Exemplary-GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCAmCmGmAmAmAmGmGmGmCmAmCmCmGmAmGmU mCmGmG*mU*mG*mC 267Exemplary- GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCAmAmAmAmUmGmGmCmAmCmCmGmAmGmUmCmG mG*mU*mG*mC 268Exemplary- GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCACmGmAmAmAmGmGmGmCmAmCmCmGmAmGmUm CmGmG*mU*mG*mC 269Exemplary- GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG mod onlyCUAGUCCGUUAUCAAmAmAmUmGmGmCmAmCmCmGmAmGmUmCmGm G*mU*mG*mC

In some embodiments, the modified sgRNA comprises the followingsequence: mN*mN*mN* NNGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 300),where “N” may be any natural or non-natural nucleotide, and wherein thetotality of N's comprise a KLKB1 guide sequence as described in Table 1.For example, encompassed herein is SEQ ID NO: 300, where the N's arereplaced with any of the guide sequences disclosed herein in Table 1(SEQ ID Nos: 1-149). Also encompassed herein are guide RNAs combiningany of the guide sequences of Table 1 (SEQ ID Nos: 1-149) combined witha conserved portion of an sgRNA, e.g. a sequence of Table 4.

Any of the modifications described below may be present in the gRNAs andmRNAs described herein.

The terms “mA,” “mC,” “mU,” or “mG” may be used to denote a nucleotidethat has been modified with 2′-O-Me.

Modification of 2′-O-methyl can be depicted as follows:

Another chemical modification that has been shown to influencenucleotide sugar rings is halogen substitution. For example, 2′-fluoro(2′-F) substitution on nucleotide sugar rings can increaseoligonucleotide binding affinity and nuclease stability.

In this application, the terms “fA,” “fC,” “fU,” or “fG” may be used todenote a nucleotide that has been substituted with 2′-F.

Substitution of 2′-F can be depicted as follows:

Phosphorothioate (PS) linkage or bond refers to a bond where a sulfur issubstituted for one nonbridging phosphate oxygen in a phosphodiesterlinkage, for example in the bonds between nucleotides bases. Whenphosphorothioates are used to generate oligonucleotides, the modifiedoligonucleotides may also be referred to as S-oligos.

A “*” may be used to depict a PS modification. In this application, theterms A*, C*, U*, or G* may be used to denote a nucleotide that islinked to the next (e.g., 3′) nucleotide with a PS bond.

In this application, the terms “mA*,” “mC*,” “mU*,” or “mG*” may be usedto denote a nucleotide that has been substituted with 2′-O-Me and thatis linked to the next (e.g., 3′) nucleotide with a PS bond.

The diagram below shows the substitution of S- into a nonbridgingphosphate oxygen, generating a PS bond in lieu of a phosphodiester bond:

Abasic nucleotides refer to those which lack nitrogenous bases. Thefigure below depicts an oligonucleotide with an abasic (also known asapurinic) site that lacks a base:

Inverted bases refer to those with linkages that are inverted from thenormal 5′ to 3′ linkage (i.e., either a 5′ to 5′ linkage or a 3′ to 3′linkage). For example:

An abasic nucleotide can be attached with an inverted linkage. Forexample, an abasic nucleotide may be attached to the terminal 5′nucleotide via a 5′ to 5′ linkage, or an abasic nucleotide may beattached to the terminal 3′ nucleotide via a 3′ to 3′ linkage. Aninverted abasic nucleotide at either the terminal 5′ or 3′ nucleotidemay also be called an inverted abasic end cap.

In some embodiments, one or more of the first three, four, or fivenucleotides at the 5′ terminus, and one or more of the last three, four,or five nucleotides at the 3′ terminus are modified. In someembodiments, the modification is a 2′-O-Me, 2′-F, inverted abasicnucleotide, PS bond, or other nucleotide modification well known in theart to increase stability and/or performance.

In some embodiments, the first four nucleotides at the 5′ terminus, andthe last four nucleotides at the 3′ terminus are linked withphosphorothioate (PS) bonds.

In some embodiments, the first three nucleotides at the 5′ terminus, andthe last three nucleotides at the 3′ terminus comprise a 2′-O-methyl(2′-O-Me) modified nucleotide. In some embodiments, the first threenucleotides at the 5′ terminus, and the last three nucleotides at the 3′terminus comprise a 2′-fluoro (2′-F) modified nucleotide. In someembodiments, the first three nucleotides at the 5′ terminus, and thelast three nucleotides at the 3′ terminus comprise an inverted abasicnucleotide.

In some embodiments, the guide RNA comprises a modified sgRNA. In someembodiments, the guide RNA comprises any conserved portion of an sgRNAshown in Table 4, optionally having a modification pattern of any of ansgRNA shown in Table 4, optionally wherein the sgRNA comprises a 5′ and3′ end modification (if not already shown in the construct of Table 4)is provided. In some embodiments, the sgRNA comprises the modificationpattern of any of an sgRNA shown in Table 4, where N is any natural ornon-natural nucleotide, and where the totality of the N's comprise aguide sequence that directs a nuclease to a target sequence in KLKB1,e.g., as shown in Table 1.

In some embodiments, the guide RNA comprises a sgRNA comprising any oneof the guide sequences of SEQ ID No: 1-149 and any conserved portion ofan sgRNA shown in Table 4, optionally having a modification pattern ofany of an sgRNA shown in Table 4, optionally wherein the sgRNA comprisesa 5′ and 3′ end modification (if not already shown in the construct ofTable 4). In some embodiments, the guide RNA comprises a sgRNAcomprising any one of the guide sequences of SEQ ID No: 1-149 and thenucleotides of SEQ ID No: 170, 171, 172, or 173, wherein the nucleotidesof SEQ ID No: 170, 171, 172, or 173 are on the 3′ end of the guidesequence, and wherein the sgRNA may be modified as shown in Table 4 orSEQ ID NO: 300.

As noted above, in some embodiments, a composition or formulationdisclosed herein comprises an mRNA comprising an open reading frame(ORF) encoding an RNA-guided DNA binding agent, such as a Cas nucleaseas described herein. In some embodiments, an mRNA comprising an ORFencoding an RNA-guided DNA binding agent, such as a Cas nuclease, isprovided, used, or administered. In some embodiments, the ORF encodingan RNA-guided DNA nuclease is a “modified RNA-guided DNA binding agentORF” or simply a “modified ORF,” which is used as shorthand to indicatethat the ORF is modified.

In some embodiments, the modified ORF may comprise a modified uridine atleast at one, a plurality of, or all uridine positions. In someembodiments, the modified uridine is a uridine modified at the 5position, e.g., with a halogen, methyl, or ethyl. In some embodiments,the modified uridine is a pseudouridine modified at the 1 position,e.g., with a halogen, methyl, or ethyl. The modified uridine can be, forexample, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine,5-iodouridine, or a combination thereof. In some embodiments, themodified uridine is 5-methoxyuridine. In some embodiments, the modifieduridine is 5-iodouridine. In some embodiments, the modified uridine ispseudouridine. In some embodiments, the modified uridine isN1-methyl-pseudouridine. In some embodiments, the modified uridine is acombination of pseudouridine and N1-methyl-pseudouridine. In someembodiments, the modified uridine is a combination of pseudouridine and5-methoxyuridine. In some embodiments, the modified uridine is acombination of N1-methyl pseudouridine and 5-methoxyuridine. In someembodiments, the modified uridine is a combination of 5-iodouridine andN1-methyl-pseudouridine. In some embodiments, the modified uridine is acombination of pseudouridine and 5-iodouridine. In some embodiments, themodified uridine is a combination of 5-iodouridine and 5-methoxyuridine.

In some embodiments, an mRNA disclosed herein comprises a 5′ cap, suchas a Cap0, Cap1, or Cap2. A 5′ cap is generally a 7-methylguanineribonucleotide (which may be further modified, as discussed below e.g.with respect to ARCA) linked through a 5′-triphosphate to the 5′position of the first nucleotide of the 5′-to-3′ chain of the mRNA,i.e., the first cap-proximal nucleotide. In Cap0, the riboses of thefirst and second cap-proximal nucleotides of the mRNA both comprise a2′-hydroxyl. In Cap1, the riboses of the first and second transcribednucleotides of the mRNA comprise a 2′-methoxy and a 2′-hydroxyl,respectively. In Cap2, the riboses of the first and second cap-proximalnucleotides of the mRNA both comprise a 2′-methoxy. See, e.g., Katibahet al. (2014) Proc Natl Acad Sci USA 111(33):12025-30; Abbas et al.(2017) Proc Natl Acad Sci USA 114(11):E2106-E2115. Most endogenoushigher eukaryotic mRNAs, including mammalian mRNAs such as human mRNAs,comprise Cap1 or Cap2. Cap0 and other cap structures differing from Cap1and Cap2 may be immunogenic in mammals, such as humans, due torecognition as “non-self” by components of the innate immune system suchas IFIT-1 and IFIT-5, which can result in elevated cytokine levelsincluding type I interferon. Components of the innate immune system suchas IFIT-1 and IFIT-5 may also compete with eIF4E for binding of an mRNAwith a cap other than Cap1 or Cap2, potentially inhibiting translationof the mRNA.

A cap can be included co-transcriptionally. For example, ARCA(anti-reverse cap analog; Thermo Fisher Scientific Cat. No. AM8045) is acap analog comprising a 7-methylguanine 3′-methoxy-5′-triphosphatelinked to the 5′ position of a guanine ribonucleotide which can beincorporated in vitro into a transcript at initiation. ARCA results in aCap0 cap in which the 2′ position of the first cap-proximal nucleotideis hydroxyl. See, e.g., Stepinski et al., (2001) “Synthesis andproperties of mRNAs containing the novel ‘anti-reverse’ cap analogs7-methyl(3′-O-methyl)GpppG and 7-methyl(3′deoxy)GpppG,” RNA 7:1486-1495. The ARCA structure is shown below.

CleanCap™ AG (m7G(5′)ppp(5′)(2′OMeA)pG; TriLink Biotechnologies Cat. No.N-7113) or CleanCap™ GG (m7G(5′)ppp(5′)(2′OMeG)pG; TriLinkBiotechnologies Cat. No. N-7133) can be used to provide a Cap1 structureco-transcriptionally. 3′-O-methylated versions of CleanCap™ AG andCleanCap™ GG are also available from TriLink Biotechnologies as Cat.Nos. N-7413 and N-7433, respectively. The CleanCap™ AG structure isshown below.

Alternatively, a cap can be added to an RNA post-transcriptionally. Forexample, Vaccinia capping enzyme is commercially available (New EnglandBiolabs Cat. No. M2080S) and has RNA triphosphatase andguanylyltransferase activities, provided by its D1 subunit, and guaninemethyltransferase, provided by its D12 subunit. As such, it can add a7-methylguanine to an RNA, so as to give Cap0, in the presence ofS-adenosyl methionine and GTP. See, e.g., Guo, P. and Moss, B. (1990)Proc. Natl. Acad. Sci. USA 87, 4023-4027; Mao, X. and Shuman, S. (1994)J. Biol. Chem. 269, 24472-24479.

In some embodiments, the mRNA further comprises a poly-adenylated(poly-A) tail. In some embodiments, the poly-A tail comprises at least20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300adenines. In some embodiments, the poly-A tail comprises 95, 96, 97, 98,99, or 100 adenine nucleotides.

D. Ribonucleoprotein Complex

In some embodiments, the disclosure provides compositions comprising oneor more gRNAs comprising one or more guide sequences from Table 1 or 2and an RNA-guided DNA binding agent, e.g., a nuclease, such as a Casnuclease, such as Cas9. In some embodiments, the RNA-guided DNA-bindingagent has cleavase activity, which can also be referred to asdouble-strand endonuclease activity. In some embodiments, the RNA-guidedDNA-binding agent comprises a Cas nuclease. Examples of Cas9 nucleasesinclude those of the type II CRISPR systems of S. pyogenes, S. aureus,and other prokaryotes (see, e.g., the list in the next paragraph), andmodified (e.g., engineered or mutant) versions thereof. See, e.g.,US2016/0312198 A1; US 2016/0312199 A1. Other examples of Cas nucleasesinclude a Csm or Cmr complex of a type III CRISPR system or the Cas10,Csm1, or Cmr2 subunit thereof; and a Cascade complex of a type I CRISPRsystem, or the Cas3 subunit thereof. In some embodiments, the Casnuclease may be from a Type-IIA, Type-IIB, or Type-IIC system. Fordiscussion of various CRISPR systems and Cas nucleases see, e.g.,Makarova et al., NAT. REV. MICROBIOL. 9:467-477 (2011); Makarova et al.,NAT. REV. MICROBIOL, 13: 722-36 (2015); Shmakov et al., MOLECULAR CELL.60:385-397 (2015).

Non-limiting exemplary species that the Cas nuclease can be derived frominclude Streptococcus pyogenes, Streptococcus thermophilus,Streptococcus sp., Staphylococcus aureus, Listeria innocua,Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes,Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis.Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene,Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomycespristinaespiralis, Streptomyces viridochromogenes, Streptomycesviridochromogenes, Streptosporangium roseum, Streptosporangium roseum.Alicyclobacillus acidocaldarius. Bacillus pseudomycoides, Bacillusselenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii,Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola,Microscilla marina, Burkholderiales bacterium, Polaromonasnaphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothecesp., Microcystis aeruginosa, Synechococcus sp., Acetohalobiumarabaticum, Ammonfex degensii, Caldicelulosiruptor becscii, CandidatusDesulforudis, Clostridium botulinum, Clostridium dificile, Finegoldiamagna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum,Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatiumvinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcuswatsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer,Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena,Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp.,Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotogamobilis, Thermosipho africanus. Streptococcus pasteurianus, Neisseriacinerea. Campylobacter lari, Parvibaculum lavamentivorans,Corynebacterium diphtheria, Acidaminococcus sp., Lachnospiraceaebacterium ND2006, and Acaryochloris marina.

In some embodiments, the Cas nuclease is the Cas9 nuclease fromStreptococcus pyogenes. In some embodiments, the Cas nuclease is theCas9 nuclease from Streptococcus thermophilus. In some embodiments, theCas nuclease is the Cas9 nuclease from Neisseria meningitidis. In someembodiments, the Cas nuclease is the Cas9 nuclease is fromStaphylococcus aureus. In some embodiments, the Cas nuclease is the Cpf1nuclease from Francisella novicida. In some embodiments, the Casnuclease is the Cpf1 nuclease from Acidaminococcus sp. In someembodiments, the Cas nuclease is the Cpf1 nuclease from Lachnospiraceaebacterium ND2006. In further embodiments, the Cas nuclease is the Cpf1nuclease from Francisella tularensis, Lachnospiraceae bacterium,Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteriabacterium, Smithella, Acidaminococcus, Candidatus Methanoplasmatermitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai,Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonasmacacae. In certain embodiments, the Cas nuclease is a Cpf1 nucleasefrom an Acidaminococcus or Lachnospiraceae.

In some embodiments, the gRNA together with an RNA-guided DNA bindingagent is called a ribonucleoprotein complex (RNP). In some embodiments,the RNA-guided DNA binding agent is a Cas nuclease. In some embodiments,the gRNA together with a Cas nuclease is called a Cas RNP. In someembodiments, the RNP comprises Type-I, Type-II, or Type-III components.In some embodiments, the Cas nuclease is the Cas9 protein from theType-II CRISPR/Cas system. In some embodiment, the gRNA together withCas9 is called a Cas9 RNP.

Wild type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domaincleaves the non-target DNA strand, and the HNH domain cleaves the targetstrand of DNA. In some embodiments, the Cas9 protein comprises more thanone RuvC domain and/or more than one HNH domain. In some embodiments,the Cas9 protein is a wild type Cas9. In each of the composition, use,and method embodiments, the Cas induces a double strand break in targetDNA.

In some embodiments, chimeric Cas nucleases are used, where one domainor region of the protein is replaced by a portion of a differentprotein. In some embodiments, a Cas nuclease domain may be replaced witha domain from a different nuclease such as FokI. In some embodiments, aCas nuclease may be a modified nuclease.

In other embodiments, the Cas nuclease may be from a Type-I CRISPR/Cassystem. In some embodiments, the Cas nuclease may be a component of theCascade complex of a Type-I CRISPR/Cas system. In some embodiments, theCas nuclease may be a Cas3 protein. In some embodiments, the Casnuclease may be from a Type-III CRISPR/Cas system. In some embodiments,the Cas nuclease may have an RNA cleavage activity.

In some embodiments, the RNA-guided DNA-binding agent has single-strandnickase activity, i.e., can cut one DNA strand to produce asingle-strand break, also known as a “nick.” In some embodiments, theRNA-guided DNA-binding agent comprises a Cas nickase. A nickase is anenzyme that creates a nick in dsDNA, i.e., cuts one strand but not theother of the DNA double helix. In some embodiments, a Cas nickase is aversion of a Cas nuclease (e.g., a Cas nuclease discussed above) inwhich an endonucleolytic active site is inactivated, e.g., by one ormore alterations (e.g., point mutations) in a catalytic domain. See,e.g., U.S. Pat. No. 8,889,356 for discussion of Cas nickases andexemplary catalytic domain alterations. In some embodiments, a Casnickase such as a Cas9 nickase has an inactivated RuvC or HNH domain.

In some embodiments, the RNA-guided DNA-binding agent is modified tocontain only one functional nuclease domain. For example, the agentprotein may be modified such that one of the nuclease domains is mutatedor fully or partially deleted to reduce its nucleic acid cleavageactivity. In some embodiments, a nickase is used having a RuvC domainwith reduced activity. In some embodiments, a nickase is used having aninactive RuvC domain. In some embodiments, a nickase is used having anHNH domain with reduced activity. In some embodiments, a nickase is usedhaving an inactive HNH domain.

In some embodiments, a conserved amino acid within a Cas proteinnuclease domain is substituted to reduce or alter nuclease activity. Insome embodiments, a Cas nuclease may comprise an amino acid substitutionin the RuvC or RuvC-like nuclease domain. Exemplary amino acidsubstitutions in the RuvC or RuvC-like nuclease domain include D10A(based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al.(2015) Cell Oct 22:163(3): 759-771. In some embodiments, the Casnuclease may comprise an amino acid substitution in the HNH or HNH-likenuclease domain. Exemplary amino acid substitutions in the HNH orHNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A(based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al.(2015). Further exemplary amino acid substitutions include D917A,E1006A, and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1)sequence (UniProtKB—AOQ7Q2 (CPF 1_FRATN)).

In some embodiments, an mRNA encoding a nickase is provided incombination with a pair of guide RNAs that are complementary to thesense and antisense strands of the target sequence, respectively. Inthis embodiment, the guide RNAs direct the nickase to a target sequenceand introduce a DSB by generating a nick on opposite strands of thetarget sequence (i.e., double nicking). In some embodiments, use ofdouble nicking may improve specificity and reduce off-target effects. Insome embodiments, a nickase is used together with two separate guideRNAs targeting opposite strands of DNA to produce a double nick in thetarget DNA. In some embodiments, a nickase is used together with twoseparate guide RNAs that are selected to be in close proximity toproduce a double nick in the target DNA.

In some embodiments, the RNA-guided DNA-binding agent lacks cleavase andnickase activity. In some embodiments, the RNA-guided DNA-binding agentcomprises a dCas DNA-binding polypeptide. A dCas polypeptide hasDNA-binding activity while essentially lacking catalytic(cleavase/nickase) activity. In some embodiments, the dCas polypeptideis a dCas9 polypeptide. In some embodiments, the RNA-guided DNA-bindingagent lacking cleavase and nickase activity or the dCas DNA-bindingpolypeptide is a version of a Cas nuclease (e.g., a Cas nucleasediscussed above) in which its endonucleolytic active sites areinactivated, e.g., by one or more alterations (e.g., point mutations) inits catalytic domains. See, e.g., US 2014/0186958 A1; US 2015/0166980A1.

In some embodiments, the RNA-guided DNA-binding agent comprises one ormore heterologous functional domains (e.g., is or comprises a fusionpolypeptide).

In some embodiments, the heterologous functional domain may facilitatetransport of the RNA-guided DNA-binding agent into the nucleus of acell. For example, the heterologous functional domain may be a nuclearlocalization signal (NLS). In some embodiments, the RNA-guidedDNA-binding agent may be fused with 1-10 NLS(s). In some embodiments,the RNA-guided DNA-binding agent may be fused with 1-5 NLS(s). In someembodiments, the RNA-guided DNA-binding agent may be fused with one NLS.Where one NLS is used, the NLS may be linked at the N-terminus or theC-terminus of the RNA-guided DNA-binding agent sequence. It may also beinserted within the RNA-guided DNA binding agent sequence. In otherembodiments, the RNA-guided DNA-binding agent may be fused with morethan one NLS. In some embodiments, the RNA-guided DNA-binding agent maybe fused with 2, 3, 4, or 5 NLSs. In some embodiments, the RNA-guidedDNA-binding agent may be fused with two NLSs. In certain circumstances,the two NLSs may be the same (e.g., two SV40 NLSs) or different. In someembodiments, the RNA-guided DNA-binding agent is fused to two SV40 NLSsequences linked at the carboxy terminus. In some embodiments, theRNA-guided DNA-binding agent may be fused with two NLSs, one linked atthe N-terminus and one at the C-terminus. In some embodiments, theRNA-guided DNA-binding agent may be fused with 3 NLSs. In someembodiments, the RNA-guided DNA-binding agent may be fused with no NLS.In some embodiments, the NLS may be a monopartite sequence, such as,e.g., the SV40 NLS, PKKKRKV (SEQ ID NO: 600) or PKKKRRV (SEQ ID NO:601). In some embodiments, the NLS may be a bipartite sequence, such asthe NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 602). In aspecific embodiment, a single PKKKRKV (SEQ ID NO: 600) NLS may be linkedat the C-terminus of the RNA-guided DNA-binding agent. One or morelinkers are optionally included at the fusion site.

In some embodiments, the heterologous functional domain may be capableof modifying the intracellular half-life of the RNA-guided DNA bindingagent. In some embodiments, the half-life of the RNA-guided DNA bindingagent may be increased. In some embodiments, the half-life of theRNA-guided DNA-binding agent may be reduced. In some embodiments, theheterologous functional domain may be capable of increasing thestability of the RNA-guided DNA-binding agent. In some embodiments, theheterologous functional domain may be capable of reducing the stabilityof the RNA-guided DNA-binding agent. In some embodiments, theheterologous functional domain may act as a signal peptide for proteindegradation. In some embodiments, the protein degradation may bemediated by proteolytic enzymes, such as, for example, proteasomes,lysosomal proteases, or calpain proteases. In some embodiments, theheterologous functional domain may comprise a PEST sequence. In someembodiments, the RNA-guided DNA-binding agent may be modified byaddition of ubiquitin or a polyubiquitin chain. In some embodiments, theubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examplesof ubiquitin-like proteins include small ubiquitin-like modifier (SUMO),ubiquitin cross-reactive protein (UCRP, also known asinterferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1(URM1), neuronal-precursor-cell-expressed developmentally downregulatedprotein-8 (NEDD8, also called Rub1 in S. cerevisiae), human leukocyteantigen F-associated (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fauubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitinfold-modifier-1 (UFM1), and ubiquitin-like protein-5 (UBL5).

In some embodiments, the heterologous functional domain may be a markerdomain. Non-limiting examples of marker domains include fluorescentproteins, purification tags, epitope tags, and reporter gene sequences.In some embodiments, the marker domain may be a fluorescent protein.Non-limiting examples of suitable fluorescent proteins include greenfluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP,Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1),yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet,PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2,Azurite, mKalama1, GFPuv, Sapphire, T-sapphire), cyan fluorescentproteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), redfluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer,mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem,HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orangefluorescent proteins (mOrange, mKO, Kusabira-Orange, MonomericKusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescentprotein. In other embodiments, the marker domain may be a purificationtag and/or an epitope tag. Non-limiting exemplary tags includeglutathione-S-transferase (GST), chitin binding protein (CBP), maltosebinding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinitypurification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus,Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, Si, T7, V5, VSV-G,6×His, 8×His, biotin carboxyl carrier protein (BCCP), poly-His, andcalmodulin. Non-limiting exemplary reporter genes includeglutathione-S-transferase (GST), horseradish peroxidase (HRP),chloramphenicol acetyltransferase (CAT), beta-galactosidase,beta-glucuronidase, luciferase, or fluorescent proteins.

In additional embodiments, the heterologous functional domain may targetthe RNA-guided DNA-binding agent to a specific organelle, cell type,tissue, or organ. In some embodiments, the heterologous functionaldomain may target the RNA-guided DNA-binding agent to mitochondria.

In further embodiments, the heterologous functional domain may be aneffector domain. When the RNA-guided DNA-binding agent is directed toits target sequence, e.g., when a Cas nuclease is directed to a targetsequence by a gRNA, the effector domain may modify or affect the targetsequence. In some embodiments, the effector domain may be chosen from anucleic acid binding domain, a nuclease domain (e.g., a non-Cas nucleasedomain), an epigenetic modification domain, a transcriptional activationdomain, or a transcriptional repressor domain. In some embodiments, theheterologous functional domain is a nuclease, such as a FokI nuclease.See, e.g., U.S. Pat. No. 9,023,649. In some embodiments, theheterologous functional domain is a transcriptional activator orrepressor. See, e.g., Qi et al., “Repurposing CRISPR as an RNA-guidedplatform for sequence-specific control of gene expression,” Cell152:1173-83 (2013); Perez-Pinera et al., “RNA-guided gene activation byCRISPR-Cas9-based transcription factors,” Nat. Methods 10:973-6 (2013);Mali et al., “CAS9 transcriptional activators for target specificityscreening and paired nickases for cooperative genome engineering,” Nat.Biotechnol. 31:833-8 (2013); Gilbert et al., “CRISPR-mediated modularRNA-guided regulation of transcription in eukaryotes,” Cell 154:442-51(2013). As such, the RNA-guided DNA-binding agent essentially becomes atranscription factor that can be directed to bind a desired targetsequence using a guide RNA.

E. Determination of Efficacy of gRNAs

In some embodiments, the efficacy of a gRNA is determined when deliveredor expressed together with other components forming an RNP. In someembodiments, the gRNA is expressed together with an RNA-guided DNAbinding agent, such as a Cas protein, e.g., Cas9. In some embodiments,the gRNA is delivered to or expressed in a cell line that already stablyexpresses an RNA-guided DNA nuclease, such as a Cas nuclease or nickase,e.g., Cas9 nuclease or nickase. In some embodiments the gRNA isdelivered to a cell as part of an RNP. In some embodiments, the gRNA isdelivered to a cell along with a mRNA encoding an RNA-guided DNAnuclease, such as a Cas nuclease or nickase, e.g., Cas9 nuclease ornickase.

As described herein, use of an RNA-guided DNA nuclease and a guide RNAdisclosed herein can lead to double-stranded breaks (DSB), single-strandbreak, and/or site-specific binding that results in nucleic acidmodification in the DNA which can produce errors in the form ofinsertion/deletion (indel) mutations upon repair by cellular machinery.Many mutations due to indels alter the reading frame or introducepremature stop codons and, therefore, produce a non-functional protein.

In some embodiments, the efficacy of particular gRNAs is determinedbased on in vitro models. In some embodiments, the in vitro model isHEK293 cells stably expressing Cas9 (HEK293_Cas9). In some embodiments,the in vitro model is HUH7 human hepatocarcinoma cells. In someembodiments, the in vitro model is HepG2 cells. In some embodiments, thein vitro model is primary human hepatocytes. In some embodiments, the invitro model is primary cynomolgus hepatocytes. With respect to usingprimary human hepatocytes, commercially available primary humanhepatocytes can be used to provide greater consistency betweenexperiments. In some embodiments, the number of off-target sites atwhich a deletion or insertion occurs in an in vitro model (e.g., inprimary human hepatocytes) is determined, e.g., by analyzing genomic DNAfrom primary human hepatocytes transfected in vitro with Cas9 mRNA andthe guide RNA. In some embodiments, such a determination comprisesanalyzing genomic DNA from primary human hepatocytes transfected invitro with Cas9 mRNA, the guide RNA, and a donor oligonucleotide.Exemplary procedures for such determinations are provided in the workingexamples below.

In some embodiments, the efficacy of particular gRNAs is determinedacross multiple in vitro cell models for a gRNA selection process. Insome embodiments, a cell line comparison of data with selected gRNAs isperformed. In some embodiments, cross screening in multiple cell modelsis performed. In some embodiments, the efficacy of particular gRNAs isdetermined in PHH or PCH for a gRNA selection process.

In some embodiments, the efficacy of particular gRNAs is determinedbased on in vivo models. In some embodiments, the in vivo model is arodent model. In some embodiments, the rodent model is a mouse whichexpresses a KLKB1 gene. In some embodiments, the rodent model is a mousewhich expresses a human KLKB1 gene. In some embodiments, the in vivomodel is a non-human primate, for example cynomolgus monkey.

In some embodiments, the efficacy of a guide RNA is measured by percentediting of KLKB1. Indel percentage can be calculated from NGSsequencing. In some embodiments, the percent editing of KLKB1 iscompared to the percent editing necessary to achieve knockdown ofprekallikrein and/or kallikrein protein, e.g., from cell culture mediaor cell lysates in the case of an in vitro model or plasma containingcirculating levels in the case of an in vivo model.

In some embodiments, the efficacy of a guide RNA is measured by thenumber and/or frequency of indels at off-target sequences within thegenome of the target cell type. In some embodiments, efficacious guideRNAs are provided which produce indels at off-target sites at very lowfrequencies (e.g., <5%) in a cell population and/or relative to thefrequency of indel creation at the target site. Thus, the disclosureprovides for guide RNAs which do not exhibit off-target indel formationin the target cell type (e.g., a hepatocyte such as PHH), or whichproduce a frequency of off-target indel formation of <5% in a cellpopulation and/or relative to the frequency of indel creation at thetarget site. In some embodiments, the disclosure provides guide RNAswhich do not exhibit any off-target indel formation in the target celltype (e.g., hepatocyte). In some embodiments, guide RNAs are providedwhich produce indels at less than 5 off-target sites, e.g., as evaluatedby one or more methods described herein. In some embodiments, guide RNAsare provided which produce indels at less than or equal to 4, 3, 2, or 1off-target site(s) e.g., as evaluated by one or more methods describedherein. In some embodiments, the off-target site(s) does not occur in aprotein coding region in the target cell (e.g., hepatocyte) genome.

In some embodiments, linear amplification is used to detect gene editingevents, such as the formation of insertion/deletion (“indel”) mutations,translocations, and homology directed repair (HDR) events in target DNA.For example, linear amplification with a unique sequence-tagged primerand isolating the tagged amplification products (herein after referredto as “UnIT,” or “Unique Identifier Tagmentation” method) may be used.

In some embodiments, the efficacy of a guide RNA is determined bymeasuring levels of KLKB1, pKal, total KLKB1 (prekallikrein+pKal), KLKB1activity, HMWK, HMWK activity, and/or bradykinin, in a sample such as abody fluid, e.g., serum, plasma, or blood.

In some embodiments, the efficacy of a guide RNA is determined bymeasuring KLKB1 mRNA levels. A decrease in KLKB1 mRNA levels isindicative of an effective guide RNA.

In some embodiments, the efficacy of a guide RNA is determined bymeasuring levels of bradykinin in a sample such as a body fluid, e.g.,serum, plasma, or blood.

In some embodiments, the efficacy of a guide RNA is determined bymeasuring levels of bradykinin and/or its degradation products in asample. In some embodiments, the efficacy of a guide RNA is determinedby measuring levels of bradykinin and/or its degradation products in theserum or plasma. A decrease in the levels of bradykinin and/or itsdegradation products in the serum or plasma is indicative of aneffective guide RNA.

One method to detect bradykinin in circulating blood is provided inFerreira, et al., Br. J. Pharmac. Chemother. (1967), 29, 367-377.Bradykinin may also be detected by an enzyme-linked immunosorbent assay(ELISA) assay with cell culture media or serum or plasma. (See, e.g.,Abcam Cat. No. ab136936; Markit-M Bradykinin (Gentaur)). In someembodiments, levels of bradykinin are measured in the same in vitro orin vivo systems or models used to measure editing. In some embodiments,levels of bradykinin are measured in cells, e.g., primary humanhepatocytes. In some embodiments, levels of bradykinin are measured in afluid such as serum or plasma. In some embodiments circulating levels ofbradykinin are measured.

In some embodiments, the efficacy of a guide RNA is determined bymeasuring levels of total kallikrein (prekallikrein and plasmakallikrein (pKal)) in a sample. In some embodiments, the efficacy of aguide RNA is determined by measuring levels of total kallikrein in asample such as a body fluid, e.g., serum, plasma, or blood. In someembodiments, the efficacy of a guide RNA is determined by measuringlevels of total kallikrein in the serum or plasma. A decrease in thelevels of total kallikrein in the serum or plasma is indicative of aneffective guide RNA. In some embodiments, serum and/or plasma totalkallikrein is decreased below 40% of basal levels. In some embodiments,levels of total kallikrein are measured using an enzyme-linkedimmunosorbent assay (ELISA) assay with cell culture media or serum orplasma. In some embodiments, levels of total kallikrein are measured inthe same in vitro or in vivo systems or models used to measure editing.In some embodiments, levels of total kallikrein are measured in cells,e.g., primary human hepatocytes. In some embodiments, levels of totalkallikrein are measured in PHH and PCH cells.

In some embodiments, the efficacy of a guide RNA is determined bymeasuring levels of prekallikrein and/or kallikrein in a sample such asa body fluid, e.g., serum, plasma, or blood. In some embodiments, theefficacy of a guide RNA is determined by measuring levels ofprekallikrein and/or kallikrein in the serum or plasma. A decrease inthe levels of prekallikrein and/or kallikrein in the serum or plasma isindicative of an effective guide RNA. In some embodiments, levels ofprekallikrein and/or kallikrein are measured using an enzyme-linkedimmunosorbent assay (ELISA) assay with cell culture media or serum orplasma. In some embodiments, levels of prekallikrein and/or kallikreinare measured in the in vitro or in vivo systems or models used tomeasure editing. In some embodiments, levels of prekallikrein and/orkallikrein are measured in cells, e.g., primary human hepatocytes, inplasma, or in cell culture media. In some embodiments, levels ofprekallikrein and/or kallikrein are measured from a plasma sample. Insome embodiments, levels of prekallikrein and/or kallikrein are measuredfrom a serum sample. Prekallikrein and/or pKal protein levels areoptionally measured by ELISA after an activation step to convertprekallikrein to its active form, pKal.

In some embodiments, the efficacy of a guide RNA is determined bymeasuring levels of prekallikrein in a sample. In some embodiments, theefficacy of a guide RNA is determined by measuring levels ofprekallikrein in a sample such as a body fluid, e.g., serum, plasma, orblood. In some embodiments, the efficacy of a guide RNA is determined bymeasuring levels of prekallikrein in the serum or plasma. A decrease inthe levels of prekallikrein in the serum or plasma is indicative of aneffective guide RNA. In some embodiments, serum and/or plasmaprekallikrein is reduced at least 60%, 70%, 80%, 85%, 90%, 95% or more.In some embodiments, serum and/or plasma total kallikrein, prekallikreinand/or kallikrein is decreased by about 60-80%, 60-90%, 60-95%, 60-100%,85-95%, or 85-100%. In some embodiments, levels of prekallikrein aremeasured using an enzyme-linked immunosorbent assay (ELISA) assay withcell culture media or serum or plasma. In some embodiments, levels ofprekallikrein are measured in the in vitro or in vivo systems or modelsused to measure editing. In some embodiments, levels of prekallikreinare measured in cells, e.g., primary human hepatocytes, in plasma, or incell culture media. In some embodiments, levels of prekallikrein aremeasured from a plasma sample. In some embodiments, levels ofprekallikrein are measured from a serum sample.

In some embodiments, the efficacy of a guide RNA is determined bymeasuring levels of pKal in a sample. In some embodiments, the efficacyof a guide RNA is determined by measuring levels of pKal in the serum orplasma. A decrease in the level of pKal in the serum or plasma isindicative of an effective guide RNA. In some embodiments, level of pKalis reduced at least 60%, 70%, 80%, 85%, 90%, 95% or more. In someembodiments, serum and/or plasma pKal is decreased by about 60-80%,60-90%, 60-95%, 60-100%, 85-95%, or 85-100%. In some embodiments, levelsof pKal are measured using an enzyme-linked immunosorbent assay (ELISA)assay with cell culture media or serum or plasma. In some embodiments,levels of pKal are measured in the in vitro or in vivo systems or modelsused to measure editing. In some embodiments, levels of pKal aremeasured in cells, e.g., primary human hepatocytes, in plasma, or incell culture media. In some embodiments, levels of pKal are measuredfrom a plasma sample. In some embodiments, levels of pKal are measuredfrom a serum sample.

In some embodiments, the efficacy of a guide RNA is determined bymeasuring levels of circulating cleaved HMWK (cHMWK) and total HMWK incitrated serum or citrated plasma. In some embodiments, the efficacy ofa guide RNA is determined by measuring levels of circulating cleavedHMWK (cHMWK) and total HMWK in the serum or plasma. A decrease in theproportion of cleaved HMWK compared to total HMWK is indicative of aneffective guide RNA. In some embodiments, the proportion of cleaved HMWKcompared to total HMWK can target a ratio of circulating plasma cHMWK tototal HMWK of less than about 60%. In some embodiments the ratio ofcHMWK to HMWK is less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%,or more. In some embodiments, levels of prekallikrein are measured usingwestern Blotting assay with cell culture media or serum or plasma. Insome embodiments, levels of cHMWK and total HMWK are measured in the invitro or in vivo systems or models used to measure editing. In someembodiments, levels of cHMWK and total HMWK are measured in cells, e.g.,primary human hepatocytes, in plasma, or in cell culture media. In someembodiments, levels of cHMWK and total HMWK are measured from a plasmasample. In some embodiments, levels of cHMWK and total HMWK are measuredfrom a serum sample.

In some embodiments, the efficacy of a guide RNA is determined bymeasuring pKal activity in a sample. A decrease in the pKal activity isindicative of an effective guide RNA. In some embodiments, the efficacyof a guide RNA is determined by measuring pKal activity in the serum orplasma.

In some embodiments, the pKal activity is measured as the capacity of acitrated serum sample or citrated plasma sample to convert HMWK to cHMWK(See Banerji et al, N Engl J Med 2017; 376:717-28). A decrease in thefinal proportion of cHMWK to total HMWK indicates a decrease in pKalactivity. The levels of cHMWK and full length HMWK can be measured bywestern blotting. In other embodiments, pKal activity is measured as thecapacity of a citrated serum sample or citrated plasma sample toenzymatically cleave a HWMK-like peptide substrate, in which case adecrease in substrate cleavage indicates a decrease in pKal activity.

In some embodiments, the pKal activity is reduced by at least 40%, 50%,60%, 70%, 80%, 85%, 90%, 95% or more. In some embodiments, the pKalactivity is decreased by about 60-80%, 60-90%, 60-95%, 60-100%, 85-95%,or 85-100%. In some embodiments, pKal activity is reduced to less thanabout 40% of basal levels. In some embodiments, pKal activity is reducedto about 40-50% of basal levels. In some embodiments, pKal activity isreduced to 20-40 or 20-50% of basal levels. In some embodiments, levelsof pKal activity are measured in the in vitro or in vivo systems ormodels used to measure editing. In some embodiments, levels of pKalactivity are measured in cells, e.g., primary human hepatocytes, inplasma, or in cell culture media. In some embodiments, levels of pKalactivity are measured from a plasma sample. In some embodiments, levelsof pKal are measured from a serum sample.

III. Therapeutic Methods

The gRNAs and associated methods and compositions disclosed herein areuseful in treating and preventing HAE and preventing symptoms of HAE. Insome embodiments, the gRNAs and associated methods and compositions areuseful for reducing the frequency of HAE attacks. In some embodiments,the gRNAs and associated methods and compositions are useful forpreventing HAE attacks. In some embodiments, the gRNAs disclosed hereinare useful in treating and preventing bradykinin production andaccumulation, bradykinin-induced swelling, angioedema obstruction of theairway, or asphyxiation. In some embodiments, the gRNAs disclosed hereinare useful in treating or preventing angioedema and attacks caused byHAE. In some embodiments, the gRNAs disclosed herein are useful forreducing the frequency of angioedema attacks, such as HAE attacks. Insome embodiments, the gRNAs disclosed herein are useful for reducing theseverity of angioedema attacks. In some embodiments, the gRNAs disclosedherein are useful for reducing the frequency and/or severity of attacks,such as HAE attacks. In some embodiments, the gRNAs disclosed herein areuseful for achieving remission of angioedema attacks, such as HAEattacks. In some embodiments, the gRNAs disclosed herein are useful forachieving durable remission, e.g. maintained for at least 1 month, 2months, 4 months, 6 months, 1 year, 2 years, 5 years, 10 years or more.

The gRNAs and associated methods and compositions disclosed herein areuseful to decrease KLKB1 mRNA production. Therefore, in one aspect,effectiveness of treatment/prevention can be assessed by measuring KLKB1mRNA levels, wherein a decrease in KLKB1 mRNA levels indicateseffectiveness.

The gRNAs and associated methods and compositions disclosed herein areuseful to decrease prekallikrein protein levels in plasma or serum.Therefore, in one aspect, effectiveness of treatment/prevention can beassessed by measuring prekallikrein protein levels or total kallikreinprotein levels, wherein a decrease in prekallikrein and/or kallikreinprotein indicates effectiveness. In some embodiments, effectiveness oftreatment/prevention can be assessed by measuring prekallikrein proteinin a sample, such as serum or plasma, wherein a decrease inprekallikrein indicates effectiveness. For example, plasma or serumprekallikrein can be measured by ELISA as described in Ferrone J D,Bhattacharjee G, Revenko A S, et al. IONIS-PKK_(Rx) a Novel AntisenseInhibitor of Prekallikrein and Bradykinin Production. Nucleic Acid Ther.2019; 29(2):82-91. Similarly, kallikrein can be measured by ELISA asdescribed herein, and administration of the gRNAs disclosed herein candecrease kallikrein protein levels in plasma or serum.

The gRNAs and associated methods and compositions disclosed herein areuseful to decrease total kallikrein (prekallikrein and pKal) proteinlevels in plasma or serum. Therefore, in one aspect, effectiveness oftreatment/prevention can be assessed by measuring total kallikrein(prekallikrein and pKal) protein levels, wherein a decrease in totalkallikrein protein indicates effectiveness. Total kallikrein,prekallikrein, and/or kallikrein may be measured before or afteractivation to release plasma kallikrein. In some embodiments,effectiveness of treatment/prevention can be assessed by measuringprekallikrein and/or pKal protein in a sample, such as serum or plasma,wherein a decrease in prekallikrein protein indicates effectiveness. Insome embodiments, effectiveness of treatment/prevention can be assessedby measuring pKal protein in a sample, such as serum or plasma, whereina decrease in pKal protein indicates effectiveness. For example, levelsof prekallikrein and pKal protein can be measured by ELISA, for exampleby using the Prekallikrein and Kallikrein Human ELISA Kit (Abcam,Eugene, OR). Prekallikrein and/or pKal protein levels are optionallymeasured by ELISA after an activation step to convert prekallikrein toits active form, pKal.

The gRNAs and associated methods and compositions disclosed herein areuseful to decrease the proportion of circulating cleaved HMWK (cHMWK)compared to total HMWK in citrated serum or citrated plasma. Therefore,in one aspect, effectiveness of treatment/prevention can be assessed bymeasuring total HMWK and cHMWK protein levels, wherein a decrease in theproportion of cleaved HMWK indicates effectiveness. In some embodiments,effectiveness of treatment/prevention can be assessed by measuring totalHMWK and cHMWK protein levels in a sample, such as serum or plasma,wherein a decrease in the proportion of cHMWK indicates effectiveness.For example, the proportion of cHMWK compared to total HMWK in citratedserum or citrated plasma samples can be measured by western blotting asdescribed in Suffritti C, Zanichelli A, Maggioni L, Bonanni E, Cugno M,Cicardi M. High-molecular weight kininogen cleavage correlates withdisease states in the bradykinin-mediated angioedema due to hereditaryC1-inhibitor deficiency. Clin Exp Allergy 2014; 44:1503-14 and inBanerji A, Busse P, Shennak M, et al. Inhibiting plasma kallikrein forhereditary angioedema prophylaxis. N Engl J Med 2017; 376:717-28.

Circulating plasma cHMWK levels below about 30% total HMWK wereassociated with decreases in HAE attacks in patients treated withlanadelumab (See Banerji, et al, 2017). In this same study, healthycontrols had plasma levels of cHMWK around 8.3% total HMWK. In anotherstudy, Suffriti and colleagues found cHMWK plasma levels of an averageof about 34.8% in normal controls, about 41.4% in HAE patients inremission and about 58.1% in HAE patients during an attack (Suffritti,et al. Clin Exp Allergy 2014; 44:1503-14). Accordingly, in someembodiments, the gRNAs and associated methods and compositions disclosedherein are useful for reducing circulating cHMWK levels such that asubject exhibits reduced number of HAE attacks. In some embodiments, thegRNAs and associated methods and compositions disclosed herein areuseful to reduce a subject's proportion of cHMWK in citrated plasma tobelow 30%. In some embodiments, the gRNAs and associated methods andcompositions disclosed herein are useful to reduce a subject'sproportion of cHMWK in citrated plasma to below 30%, 20%, and/or 10%. Insome embodiments, the gRNAs and associated methods and compositionsdisclosed herein are useful to reduce a subject's proportion of cHMWK incitrated plasma to about those of healthy controls.

The gRNAs and associated methods and compositions disclosed herein canbe useful to decrease the spontaneous pKal activity in serum or plasma.Therefore, in one aspect, effectiveness of treatment/prevention can beassessed by measuring spontaneous pKal activity, wherein a decrease inspontaneous pKal activity indicates effectiveness. In some embodiments,effectiveness of treatment/prevention can be assessed by measuringspontaneous pKal activity in a sample, such as serum or plasma, whereina decrease in spontaneous pKal activity indicates effectiveness. Incertain embodiments, the gRNAs and associated methods and compositionsdisclosed herein are useful to decrease the basal level of circulatingpKal and circulating pKal activity.

The gRNAs and associated methods and compositions disclosed herein canbe useful to decrease the inducible pKal activity in serum or plasma.Therefore, in one aspect, effectiveness of treatment/prevention can beassessed by measuring inducible pKal activity, wherein a decrease ininducible pKal activity indicates effectiveness. In some embodiments,effectiveness of treatment/prevention can be assessed by measuringinducible pKal activity in a sample, such as serum or plasma, wherein adecrease in inducible pKal activity indicates effectiveness. In someexamples, pKal activity can be induced by exposure of a sample to FXIIa(See Banerji et al, N Engl J Med 2017; 376:717-28.) In some examples,pKal activity can be induced by incubation of a sample with dextransulfate (See Ferrone, et al. Nucleic Acid Ther. 2019:29(2):82-91.) Insome examples pKal activity can be induced by addition of ellagic acidto a sample (Aygören-Pürsün, et al. J Allergy Clin Immunol 2016; 138:934-936.)

In some examples, pKal activity is measured as the capacity of acitrated serum sample or citrated plasma sample to convert HMWK to cHMWK(See Banerji et al, N Engl J Med 2017; 376:717-28) wherein a decrease inthe final proportion of cHMWK to total HMWK indicates a decrease in pKalactivity. The proportion of cHMWK and full length HMWK can be measuredby Western blotting, for instance as described by Suffritti, et al. ClinExp Allergy 2014; 44:1503-14. In other examples, pKal activity ismeasured as the capacity of a citrated serum sample or citrated plasmasample to enzymatically cleave a HWMK-like peptide substrate, in whichcase a decrease in substrate cleavage indicates a decrease in pKalactivity. In one example, the substrate peptide can be the chromogenicsubstrate H-D-Pro-Phe-Arg-p-nitroanilide peptide (Bachem, Cat. L-2120)and cleavage can be measured as change in A405 (See Defendi et al, PLoSOne 2013; 8:e70140). In another example the substrate peptide can be thefluorogenic substrate H-Pro-Phe-Arg-AMC (Sigma, Cat No. P9273) andcleavage can be measured as fluorescence changes as excitation andemission wavelengths at 360 and 480 nm, respectively (See Banerji, etal., N Engl J Med 2017; 376:717-28).

In one study, reduction of induced pKal activity by more than 40% wasassociated with a reduction in HAE attacks (Banerji, et al., N Engl JMed 2017; 376:717-28). Reduction of induced pKal activity by at least50% was associated with a reduction in HAE attacks with treatment byBCX7353 (Aygören-Pürsün, et al., N Engl J Med 2018; 379:352-362).Reductions of induced pKal activity by 60% have been associated withreduction in attacks in treatment with lanadelumab (Banerji, et al., NEngl J Med 2017; 376:717-28). Accordingly, in some embodiments,administration of the gRNAs and compositions disclosed herein are usefulfor reducing kallikrein activity, e.g. total kallikrein, prekallikrein,and/or pKal activity) such that a subject exhibits fewer HAE attacks.

In some embodiments, administration of the gRNAs and compositionsdisclosed herein reduces a subject's pKal activity to less than about40% of basal levels. In some embodiments, administration of the gRNAsand compositions disclosed herein reduces a subject's pKal activity toabout 40-50% of basal levels. In some embodiments, administration of thegRNAs and compositions disclosed herein reduces a subject's pKalactivity to 20-40% or 20-50% of basal levels.

In some embodiments, any one or more of the gRNAs, compositions, orpharmaceutical formulations described herein is for use in preparing amedicament for treating or preventing a disease or disorder in asubject. In some embodiments, treatment and/or prevention isaccomplished with a single dose, e.g., one-time treatment, ofmedicament/composition. In some embodiments, the disease or disorder isHAE.

In some embodiments, the invention comprises a method of treating orpreventing a disease or disorder in subject comprising administering anyone or more of the gRNAs, compositions, or pharmaceutical formulationsdescribed herein. In some embodiments, the disease or disorder is HAE.In some embodiments, the gRNAs, compositions, or pharmaceuticalformulations described herein are administered as a single dose, e.g.,at one time. In some embodiments, the single dose achieves durabletreatment and/or prevention. In some embodiments, the method achievesdurable treatment and/or prevention. Durable treatment and/orprevention, as used herein, includes treatment and/or prevention thatextends at least i) 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15weeks; ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, or 36months; or iii) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In someembodiments, a single dose of the gRNAs, compositions, or pharmaceuticalformulations described herein is sufficient to treat and/or prevent anyof the indications described herein for the duration of the subject'slife.

In some embodiments, the invention comprises a method or use ofmodifying (e.g., creating a double strand break) a target DNAcomprising, administering or delivering any one or more of the gRNAs,compositions, or pharmaceutical formulations described herein. In someembodiments, the target DNA is the KLKB1 gene. In some embodiments, thetarget DNA is in an exon of the KLKB1 gene. In some embodiments, thetarget DNA is in exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or15 of the KLKB1 gene.

In some embodiments, the invention comprises a method or use formodulation of a target gene comprising, administering or delivering anyone or more of the gRNAs, compositions, or pharmaceutical formulationsdescribed herein. In some embodiments, the modulation is editing of theKLKB1 target gene. In some embodiments, the modulation is a change inexpression of the protein encoded by the KLKB1 target gene.

In some embodiments, the method or use results in gene editing. In someembodiments, the method or use results in a double-stranded break withinthe target KLKB1 gene. In some embodiments, the method or use results information of indel mutations during non-homologous end joining of theDSB. In some embodiments, the method or use results in an insertion ordeletion of nucleotides in a target KLKB1 gene. In some embodiments, theinsertion or deletion of nucleotides in a target KLKB1 gene leads to aframeshift mutation or premature stop codon that results in anon-functional protein. In some embodiments, the insertion or deletionof nucleotides in a target KLKB1 gene leads to a knockdown orelimination of target gene expression.

In some embodiments, the method or use results in KLKB1 gene modulation.In some embodiments, the KLKB1 gene modulation is a decrease in geneexpression. In some embodiments, the method or use results in decreasedexpression of the protein encoded by the target gene in a population ofcells or in vivo.

In some embodiments, a method of inducing a double-stranded break (DSB)within the KLKB1 gene is provided comprising administering a compositioncomprising a guide RNA comprising any one or more guide sequences of SEQID NOs: 1-149. In some embodiments, gRNAs comprising any one or more ofthe guide sequences of SEQ ID NOs: 1-149 are administered to induce aDSB in the KLKB1 gene. The guide RNAs may be administered together withan RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or anmRNA or vector encoding an RNA-guided DNA nuclease such as a Casnuclease (e.g., Cas9).

In some embodiments, a method of modifying the KLKB1 gene is providedcomprising administering a composition comprising a guide RNA comprisingany one or more of the guide sequences of SEQ ID NOs: 1-149. In someembodiments, gRNAs comprising any one or more of the guide sequences ofSEQ ID NOs: 1-149 are administered to modify the KLKB1 gene. The guideRNAs may be administered together with an RNA-guided DNA nuclease suchas a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding anRNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).

In some embodiments, a method of treating or preventing hereditaryangioedema (HAE) is provided comprising administering a compositioncomprising a guide RNA comprising any one or more of the guide sequencesof SEQ ID NOs: 1-149. In some embodiments, gRNAs comprising any one ormore of the guide sequences of SEQ ID NOs: 1-149 are administered totreat or prevent HAE. The guide RNAs may be administered together withan RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or anmRNA or vector encoding an RNA-guided DNA nuclease such as a Casnuclease (e.g., Cas9).

In some embodiments, a method of decreasing or eliminating bradykininproduction and accumulation is provided comprising administering a guideRNA comprising any one or more of the guide sequences of SEQ ID NOs:1-149. The guide RNAs may be administered together with an RNA-guidedDNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vectorencoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).

In some embodiments, a method of treating or preventingbradykinin-induced swelling is provided comprising administering a guideRNA comprising any one or more of the guide sequences of SEQ ID NOs:1-149. The guide RNAs may be administered together with an RNA-guidedDNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vectorencoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).

In some embodiments, a method of treating or preventingbradykinin-induced angioedema is provided comprising administering aguide RNA comprising any one or more of the guide sequences of SEQ IDNOs: 1-149. The guide RNAs may be administered together with anRNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNAor vector encoding an RNA-guided DNA nuclease such as a Cas nuclease(e.g., Cas9).

In some embodiments, a method of treating or preventing obstruction ofthe airway and/or asphyxiation is provided comprising administering aguide RNA comprising any one or more of the guide sequences of SEQ IDNOs: 1-149. The guide RNAs may be administered together with anRNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNAor vector encoding an RNA-guided DNA nuclease such as a Cas nuclease(e.g., Cas9).

In some embodiments, gRNAs comprising any one or more of the guidesequences of SEQ ID NOs: 1-149 are administered to reduce bradykininlevels in the plasma, serum, or blood. The gRNAs may be administeredtogether with an RNA-guided DNA nuclease such as a Cas nuclease (e.g.,Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as aCas nuclease (e.g., Cas9).

In some embodiments, gRNAs comprising any one or more of the guidesequences of SEQ ID NOs: 1-149 are administered to decrease bradykininin the serum or plasma. The gRNAs may be administered together with anRNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNAor vector encoding an RNA-guided DNA nuclease such as a Cas nuclease(e.g., Cas9).

In some embodiments, the gRNAs comprising the guide sequences of Table 1together with an RNA-guided DNA nuclease such as a Cas nuclease induceDSBs, and non-homologous ending joining (NHEJ) during repair leads to amutation in the KLKB1 gene. In some embodiments, NHEJ leads to adeletion or insertion of a nucleotide(s), which induces a frame shift ornonsense mutation in the KLKB1 gene.

In some embodiments, administering the guide RNAs of the invention(e.g., in a composition provided herein) decrease levels (e.g., serum orplasma levels) of total kallikrein, prekallikrein, and/or kallikrein inthe subject, and therefore prevents bradykinin overproduction andaccumulation. In some embodiments, administering the guide RNAs of theinvention (e.g., in a composition provided herein) decrease kallikreinactivity levels (e.g., serum or plasma levels) in the subject, andtherefore prevents bradykinin overproduction and accumulation.

In some embodiments, the methods provided herein result in fewer attacksthat include fluid leakage through blood cells to tissues. In someembodiments, the methods provided herein reduce the frequency of attacksthat increase swelling in organ tissues. In some embodiments,administering the guide RNAs of the invention (e.g., in a compositionprovided herein) decrease the frequency or severity of angioedemaattacks.

In some embodiments, the subject is mammalian. In some embodiments, thesubject is a primate, e.g. human.

In some embodiments, the use of a guide RNAs comprising any one or moreof the guide sequences in Table 1 or Table 2 (e.g., in a compositionprovided herein) is provided for the preparation of a medicament fortreating a human subject having HAE.

In some embodiments, the guide RNAs, compositions, and formulations areadministered intravenously. In some embodiments, the guide RNAs,compositions, and formulations are administered by infusion. In someembodiments, the guide RNAs, compositions, and formulations areadministered into the hepatic circulation.

In some embodiments, a single administration of a composition comprisinga guide RNA provided herein is sufficient to knock down expression ofthe protein. In other embodiments, more than one administration of acomposition comprising a guide RNA provided herein may be beneficial tomaximize therapeutic effects.

In some embodiments, treatment slows or halts HAE disease progression.

In some embodiments, treatment slows or halts progression of angioedema.In some embodiments, treatment results in improvement, stabilization, orslowing of change in symptoms of HAE.

A. Delivery of gRNA Compositions

Lipid nanoparticles (LNPs) are a well-known means for delivery ofnucleotide and protein cargo and may be used for delivery of the guideRNAs, compositions, or pharmaceutical formulations disclosed herein. Insome embodiments, the LNPs deliver nucleic acid, protein, or nucleicacid together with protein.

In some embodiments, the invention comprises a method for delivering anyone of the gRNAs disclosed herein to a subject, wherein the gRNA isassociated with an LNP. In some embodiments, the gRNA/LNP is alsoassociated with a Cas9 or an mRNA encoding Cas9.

In some embodiments, the invention comprises a composition comprisingany one of the gRNAs disclosed and an LNP. In some embodiments, thecomposition further comprises a Cas9 or an mRNA encoding Cas9.

In some embodiments, the LNPs comprise cationic lipids. In someembodiments, the LNPs comprise(9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate, also called3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate) or another ionizable lipid. See, e.g.,lipids of WO/2017/173054 and references described therein. In someembodiments, the LNPs comprise molar ratios of a cationic lipid amine toRNA phosphate (N:P) of about 4.5, 5.0, 5.5, 6.0, or 6.5. In someembodiments, the term cationic and ionizable in the context of LNPlipids is interchangeable, e.g., wherein ionizable lipids are cationicdepending on the pH.

In some embodiments, LNPs associated with the gRNAs disclosed herein arefor use in preparing a medicament for treating a disease or disorder.

Electroporation is a well-known means for delivery of cargo, and anyelectroporation methodology may be used for delivery of any one of thegRNAs disclosed herein. In some embodiments, electroporation may be usedto deliver any one of the gRNAs disclosed herein and Cas9 or an mRNAencoding Cas9.

In some embodiments, the invention comprises a method for delivering anyone of the gRNAs disclosed herein to an ex vivo cell, wherein the gRNAis associated with an LNP or not associated with an LNP. In someembodiments, the gRNA/LNP or gRNA is also associated with a Cas9 or anmRNA encoding Cas9.

In some embodiments, the guide RNA compositions described herein, aloneor encoded on one or more vectors, are formulated in or administered viaa lipid nanoparticle; see e.g., WO/2017/173054 and WO 2019/067992, thecontents of which are hereby incorporated by reference in theirentirety.

In certain embodiments, the invention comprises DNA or RNA vectorsencoding any of the guide RNAs comprising any one or more of the guidesequences described herein. In some embodiments, in addition to guideRNA sequences, the vectors further comprise nucleic acids that do notencode guide RNAs. Nucleic acids that do not encode guide RNA include,but are not limited to, promoters, enhancers, regulatory sequences, andnucleic acids encoding an RNA-guided DNA nuclease, which can be anuclease such as Cas9. In some embodiments, the vector comprises one ormore nucleotide sequence(s) encoding a crRNA, a trRNA, or a crRNA andtrRNA. In some embodiments, the vector comprises one or more nucleotidesequence(s) encoding a sgRNA and an mRNA encoding an RNA-guided DNAnuclease, which can be a Cas nuclease, such as Cas9 or Cpf1. In someembodiments, the vector comprises one or more nucleotide sequence(s)encoding a crRNA, a trRNA, and an mRNA encoding an RNA-guided DNAnuclease, which can be a Cas protein, such as, Cas9. In one embodiment,the Cas9 is from Streptococcus pyogenes (i.e., Spy Cas9). In someembodiments, the nucleotide sequence encoding the crRNA, trRNA, or crRNAand trRNA (which may be a sgRNA) comprises or consists of a guidesequence flanked by all or a portion of a repeat sequence from anaturally-occurring CRISPR/Cas system. The nucleic acid comprising orconsisting of the crRNA, trRNA, or crRNA and trRNA may further comprisea vector sequence wherein the vector sequence comprises or consists ofnucleic acids that are not naturally found together with the crRNA,trRNA, or crRNA and trRNA.

This description and exemplary embodiments should not be taken aslimiting. For the purposes of this specification and appended claims,unless otherwise indicated, all numbers expressing quantities,percentages, or proportions, and other numerical values used in thespecification and claims, are to be understood as being modified in allinstances by the term “about,” to the extent they are not already somodified. Accordingly, unless indicated to the contrary, the numericalparameters set forth in the following specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques.

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” and any singular use of anyword, include plural referents unless expressly and unequivocallylimited to one referent. As used herein, the term “include” and itsgrammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

EXAMPLES

The following examples are provided to illustrate certain disclosedembodiments and are not to be construed as limiting the scope of thisdisclosure in any way.

Example 1: Materials and Methods

1.1 In Vitro Transcription (“IVT”) of Nuclease mRNA

Capped and polyadenylated Streptococcus pyogenes (“Spy”) Cas9 mRNAcontaining N1-methyl pseudo-U was generated by in vitro transcriptionusing a linearized plasmid DNA template and T7 RNA polymerase. PlasmidDNA containing a T7 promoter and a sequence for transcription (forproducing mRNA comprising an mRNA described herein (see SEQ ID NOs:501-516 in Table 5 below for Cas9 ORFs) was linearized by incubating at37° C. to complete digestion with XbaI with the following conditions:200 ng/μL plasmid, 2 U/μL XbaI (NEB), and 1× reaction buffer. The XbaIwas inactivated by heating the reaction at 65° C. for 20 min. Thelinearized plasmid was purified from enzyme and buffer salts using asilica maxi spin column (Epoch Life Sciences) and analyzed by agarosegel to confirm linearization. The IVT reaction to generate Cas9 mRNA wasincubated at 37° C. for 4 hours in the following conditions: 50 ng/μLlinearized plasmid; 2 mM each of GTP, ATP, CTP, and N1-methyl pseudo-UTP(Trilink); 10 mM ARCA (Trilink); 5 U/μL T7 RNA polymerase (NEB); 1 U/μLMurine RNase inhibitor (NEB); 0.004 U/μL Inorganic E. colipyrophosphatase (NEB); and 1× reaction buffer. After the 4-hourincubation, TURBO DNase (ThermoFisher) was added to a finalconcentration of 0.01 U/μL, and the reaction was incubated for anadditional 30 minutes to remove the DNA template. The Cas9 mRNA waspurified from enzyme and nucleotides using a MegaClear TranscriptionClean-up kit according to the manufacturer's protocol (ThermoFisher).Alternatively, the Cas9 mRNA was purified with a LiCl precipitationmethod, which in some cases was followed by further purification bytangential flow filtration. The transcript concentration was determinedby measuring the light absorbance at 260 nm (Nanodrop), and thetranscript was analyzed by capillary electrophoresis by Bioanlayzer(Agilent).

TABLE 5 Exemplary Cas9 mRNA Sequences SEQ ID NO Sequence 501GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTTGCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG 502AUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGAGGUCCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGGAGAAACAGGAGAACAGCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUCAGCAACGAAAUGGCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCAAAAGUACCCGACAAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUCUACCUAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUGGGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGCAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAA

GCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGA

CUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGAAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAA

GAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACAUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAA

GAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCG

AGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGAAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAAACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCCAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUGAGCCAGCUGGGAGGAGACUAG 503GACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC 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506GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTTGCAGGCCTTATTCGGATCCATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG 507ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACcggCTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAaAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAG 508ATGGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGGGCAACACCGACAGACACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCAGACTGAAGAGAACCGCCAGAAGAAGATACACCAGAAGAAAGAACAGAATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAGGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGAGACACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGAGACTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCAGAGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGAGCGCCAGACTGAGCAAGAGCAGAAGACTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGAGACAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACAGAGAGGACCTGCTGAGAAAGCAGAGAACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGAGAAGACAGGAGGACTTCTACCCCTTCCTGAAGGACAACAGAGAGAAGATCGAGAAGATCCTGACCTTCAGAATCCCCTACTACGTGGGCCCCCTGGCCAGAGGCAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGAGAATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACAGAAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGGAGGACAGATTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACAGAGAGATGATCGAGGAGAGACTGAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGAGAAGAAGATACACCGGCTGGGGCAGACTGAGCAGAAAGCTGATCAACGGCATCAGAGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCAGACACAAGCCCGAGAACATCGTGATCGAGATGGCCAGAGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCAGAGAGAGAATGAAGAGAATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCAGAGACATGTACGTGGACCAGGAGCTGGACATCAACAGACTGAGCGACTACGACGTGGACCACATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACAGAGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCCAAGCTGATCACCCAGAGAAAGTTCGACAACCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAGACCAGACAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCAGAATGAACACCAAGTACGACGAGAACGACAAGCTGATCAGAGAGGTGAAGGTGATCACCCTGAAGAGCAAGCTGGTGAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTGAGAGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGAGAAAGATGATCGCCAAGAGCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGATCAGAAAGAGACCCCTGATCGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCAGAGACTTCGCCACCGTGAGAAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGAGAAACAGCGACAAGCTGATCGCCAGAAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGAGAAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCAGAAAGAGAATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGAGAGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACAAGCCCATCAGAGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGACAGAAAGAGATACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACCAGAATCGACCTGAGCCAGCTGGGCGGCGACGGCGGCGGCAGCCCCAAGAAGAAGAGAAAGGTGTGA 509GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTTGCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGGGCAACACCGACAGACACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCAGACTGAAGAGAACCGCCAGAAGAAGATACACCAGAAGAAAGAACAGAATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAGGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGAGACACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGAGACTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCAGAGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGAGCGCCAGACTGAGCAAGAGCAGAAGACTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGAGACAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACAGAGAGGACCTGCTGAGAAAGCAGAGAACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGAGAAGACAGGAGGACTTCTACCCCTTCCTGAAGGACAACAGAGAGAAGATCGAGAAGATCCTGACCTTCAGAATCCCCTACTACGTGGGCCCCCTGGCCAGAGGCAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGAGAATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACAGAAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGGAGGACAGATTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACAGAGAGATGATCGAGGAGAGACTGAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGAGAAGAAGATACACCGGCTGGGGCAGACTGAGCAGAAAGCTGATCAACGGCATCAGAGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCAGACACAAGCCCGAGAACATCGTGATCGAGATGGCCAGAGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCAGAGAGAGAATGAAGAGAATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCAGAGACATGTACGTGGACCAGGAGCTGGACATCAACAGACTGAGCGACTACGACGTGGACCACATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACAGAGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCCAAGCTGATCACCCAGAGAAAGTTCGACAACCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAGACCAGACAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCAGAATGAACACCAAGTACGACGAGAACGACAAGCTGATCAGAGAGGTGAAGGTGATCACCCTGAAGAGCAAGCTGGTGAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTGAGAGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGAGAAAGATGATCGCCAAGAGCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGATCAGAAAGAGACCCCTGATCGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCAGAGACTTCGCCACCGTGAGAAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGAGAAACAGCGACAAGCTGATCGCCAGAAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGAGAAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCAGAAAGAGAATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGAGAGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACAAGCCCATCAGAGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGACAGAAAGAGATACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACCAGAATCGACCTGAGCCAGCTGGGCGGCGACGGCGGCGGCAGCCCCAAGAAGAAGAGAAAGGTGTGACTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG 510ATGGACAAGAAGTACTCTATCGGTTTGGACATCGGTACCAACTCTGTCGGTTGGGCCGTCATCACCGACGAATACAAGGTCCCATCTAAGAAGTTCAAGGTCTTGGGTAACACCGACAGACACTCTATCAAGAAGAACTTGATCGGTGCCTTGTTGTTCGACTCTGGTGAAACCGCCGAAGCCACCAGATTGAAGAGAACCGCCAGAAGAAGATACACCAGAAGAAAGAACAGAATCTGCTACTTGCAAGAAATCTTCTCTAACGAAATGGCCAAGGTCGACGACTCTTTCTTCCACAGATTGGAAGAATCTTTCTTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCAATCTTCGGTAACATCGTCGACGAAGTCGCCTACCACGAAAAGTACCCAACCATCTACCACTTGAGAAAGAAGTTGGTCGACTCTACCGACAAGGCCGACTTGAGATTGATCTACTTGGCCTTGGCCCACATGATCAAGTTCAGAGGTCACTTCTTGATCGAAGGTGACTTGAACCCAGACAACTCTGACGTCGACAAGTTGTTCATCCAATTGGTCCAAACCTACAACCAATTGTTCGAAGAAAACCCAATCAACGCCTCTGGTGTCGACGCCAAGGCCATCTTGTCTGCCAGATTGTCTAAGAGCAGAAGATTGGAAAACTTGATCGCCCAATTGCCAGGTGAAAAGAAGAACGGTTTGTTCGGTAACTTGATCGCCTTGTCTTTGGGTTTGACCCCAAACTTCAAGTCTAACTTCGACTTGGCCGAAGACGCCAAGTTGCAATTGTCTAAGGACACCTACGACGACGACTTGGACAACTTGTTGGCCCAAATCGGTGACCAATACGCCGACTTGTTCTTGGCCGCCAAGAACTTGTCTGACGCCATCTTGTTGTCTGACATCTTGAGAGTCAACACCGAAATCACCAAGGCCCCATTGTCTGCCTCTATGATCAAGAGATACGACGAACACCACCAAGACTTGACCTTGTTGAAGGCCTTGGTCAGACAACAATTGCCAGAAAAGTACAAGGAAATCTTCTTCGACCAATCTAAGAACGGTTACGCCGGTTACATCGACGGTGGTGCCTCTCAAGAAGAATTCTACAAGTTCATCAAGCCAATCTTGGAAAAGATGGACGGTACCGAAGAATTGTTGGTCAAGTTGAACAGAGAAGACTTGTTGAGAAAGCAAAGAACCTTCGACAACGGTTCTATCCCACACCAAATCCACTTGGGTGAATTGCACGCCATCTTGAGAAGACAAGAAGACTTCTACCCATTCTTGAAGGACAACAGAGAAAAGATCGAAAAGATCTTGACCTTCAGAATCCCATACTACGTCGGTCCATTGGCCAGAGGTAACAGCAGATTCGCCTGGATGACCAGAAAGTCTGAAGAAACCATCACCCCATGGAACTTCGAAGAAGTCGTCGACAAGGGTGCCTCTGCCCAATCTTTCATCGAAAGAATGACCAACTTCGACAAGAACTTGCCAAACGAAAAGGTCTTGCCAAAGCACTCTTTGTTGTACGAATACTTCACCGTCTACAACGAATTGACCAAGGTCAAGTACGTCACCGAAGGTATGAGAAAGCCAGCCTTCTTGTCTGGTGAACAAAAGAAGGCCATCGTCGACTTGTTGTTCAAGACCAACAGAAAGGTCACCGTCAAGCAATTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACTCTGTCGAAATCTCTGGTGTCGAAGACAGATTCAACGCCTCTTTGGGTACCTACCACGACTTGTTGAAGATCATCAAGGACAAGGACTTCTTGGACAACGAAGAAAACGAAGACATCTTGGAAGACATCGTCTTGACCTTGACCTTGTTCGAAGACAGAGAAATGATCGAAGAAAGATTGAAGACCTACGCCCACTTGTTCGACGACAAGGTCATGAAGCAATTGAAGAGAAGAAGATACACCGGTTGGGGTAGATTGAGCAGAAAGTTGATCAACGGTATCAGAGACAAGCAATCTGGTAAGACCATCTTGGACTTCTTGAAGTCTGACGGTTTCGCCAACAGAAACTTCATGCAATTGATCCACGACGACTCTTTGACCTTCAAGGAAGACATCCAAAAGGCCCAAGTCTCTGGTCAAGGTGACTCTTTGCACGAACACATCGCCAACTTGGCCGGTTCTCCAGCCATCAAGAAGGGTATCTTGCAAACCGTCAAGGTCGTCGACGAATTGGTCAAGGTCATGGGTAGACACAAGCCAGAAAACATCGTCATCGAAATGGCCAGAGAAAACCAAACCACCCAAAAGGGTCAAAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGTATCAAGGAATTGGGTTCTCAAATCTTGAAGGAACACCCAGTCGAAAACACCCAATTGCAAAACGAAAAGTTGTACTTGTACTACTTGCAAAACGGTAGAGACATGTACGTCGACCAAGAATTGGACATCAACAGATTGTCTGACTACGACGTCGACCACATCGTCCCACAATCTTTCTTGAAGGACGACTCTATCGACAACAAGGTCTTGACCAGATCTGACAAGAACAGAGGTAAGTCTGACAACGTCCCATCTGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAATTGTTGAACGCCAAGTTGATCACCCAAAGAAAGTTCGACAACTTGACCAAGGCCGAAAGAGGTGGTTTGTCTGAATTGGACAAGGCCGGTTTCATCAAGAGACAATTGGTCGAAACCAGACAAATCACCAAGCACGTCGCCCAAATCTTGGACAGCAGAATGAACACCAAGTACGACGAAAACGACAAGTTGATCAGAGAAGTCAAGGTCATCACCTTGAAGTCTAAGTTGGTCTCTGACTTCAGAAAGGACTTCCAATTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCCCACGACGCCTACTTGAACGCCGTCGTCGGTACCGCCTTGATCAAGAAGTACCCAAAGTTGGAATCTGAATTCGTCTACGGTGACTACAAGGTCTACGACGTCAGAAAGATGATCGCCAAGTCTGAACAAGAAATCGGTAAGGCCACCGCCAAGTACTTCTTCTACTCTAACATCATGAACTTCTTCAAGACCGAAATCACCTTGGCCAACGGTGAAATCAGAAAGAGACCATTGATCGAAACCAACGGTGAAACCGGTGAAATCGTCTGGGACAAGGGTAGAGACTTCGCCACCGTCAGAAAGGTCTTGTCTATGCCACAAGTCAACATCGTCAAGAAGACCGAAGTCCAAACCGGTGGTTTCTCTAAGGAATCTATCTTGCCAAAGAGAAACTCTGACAAGTTGATCGCCAGAAAGAAGGACTGGGACCCAAAGAAGTACGGTGGTTTCGACTCTCCAACCGTCGCCTACTCTGTCTTGGTCGTCGCCAAGGTCGAAAAGGGTAAGTCTAAGAAGTTGAAGTCTGTCAAGGAATTGTTGGGTATCACCATCATGGAAAGATCTTCTTTCGAAAAGAACCCAATCGACTTCTTGGAAGCCAAGGGTTACAAGGAAGTCAAGAAGGACTTGATCATCAAGTTGCCAAAGTACTCTTTGTTCGAATTGGAAAACGGTAGAAAGAGAATGTTGGCCTCTGCCGGTGAATTGCAAAAGGGTAACGAATTGGCCTTGCCATCTAAGTACGTCAACTTCTTGTACTTGGCCTCTCACTACGAAAAGTTGAAGGGTTCTCCAGAAGACAACGAACAAAAGCAATTGTTCGTCGAACAACACAAGCACTACTTGGACGAAATCATCGAACAAATCTCTGAATTCTCTAAGAGAGTCATCTTGGCCGACGCCAACTTGGACAAGGTCTTGTCTGCCTACAACAAGCACAGAGACAAGCCAATCAGAGAACAAGCCGAAAACATCATCCACTTGTTCACCTTGACCAACTTGGGTGCCCCAGCCGCCTTCAAGTACTTCGACACCACCATCGACAGAAAGAGATACACCTCTACCAAGGAAGTCTTGGACGCCACCTTGATCCACCAATCTATCACCGGTTTGTACGAAACCAGAATCGACTTGTCTCAATTGGGTGGTGACGGTGGTGGTTCTCCAAAGAAGAAGAGAAAGGTCTAA 511ATGGACAAGAAGTACTCCATCGGCCTGGACATCGGCACCAACTCCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCTCCAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGAGTCCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACTCCACCGACAAGGCCGACCTGCGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTGGACGCCAAGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAACACCGAGATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCTGGATGACCCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAGGACCGGTTCAACGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGAGCACATCGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGGCTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTGGACCAGGAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACCACATCGTGCCCCAGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCCGGTCCGACAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAAGCACGTGGCCCAGATCCTGGACTCCCGGATGAACACCAAGTACGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGCTGGTGTCCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGTCCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGACCGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTCCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACTCCCCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGTCCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGCGGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACTCCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCTGTACCTGGCCTCCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCTCCGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTGTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGTCCATCACCGGCCTGTACGAGACCCGGATCGACCTGTCCCAGCTGGGCGGCGACGGCGGCGGCTCCCCCAAGAAGAAGCGGAAGGTGTGA 512ATGGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGCCTGGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACCACATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAGCAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACGGCGGCGGCAGCCCCAAGAAGAAGCGGAAGGTGTGA 513ATGGACAAGAAGTACTCCATCGGCCTGGACATCGGCACCAACTCCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCTCCAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGAGTCCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACTCCACCGACAAGGCCGACCTGCGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTGGACGCCAAGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAACACCGAGATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCTGGATGACCCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAGGACCGGTTCAACGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGAGCACATCGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGGCTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTGGACCAGGAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACCACATCGTGCCCCAGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCCGGTCCGACAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAAGCACGTGGCCCAGATCCTGGACTCCCGGATGAACACCAAGTACGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGCTGGTGTCCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGTCCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGACCGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTCCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACTCCCCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGTCCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGCGGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACTCCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCTGTACCTGGCCTCCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCTCCGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTGTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGTCCATCACCGGCCTGTACGAGACCCGGATCGACCTGTCCCAGCTGGGCGGCGACGGCTCCGGCTCCCCCAAGAAGAAGCGGAAGGTGGACGGCTCCCCCAAGAAGAAGCGGAAGGTGGACTCCGGCTGA514ATGGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGCCTGGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACCACATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAGCAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACGGCAGCGGCAGCCCCAAGAAGAAGCGGAAGGTGGACGGCAGCCCCAAGAAGAAGCGGAAGGTGGACAGCGGCTGA 515ATGGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGCCTGGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACCACATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAGCAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACTGA 516GGGAAGCUCAGAAUAAACGCUCAACUUUGGCCGGAUCUGCCACCAUGGACAAGAAGUACUCCAUCGGCCUGGACAUCGGCACCAACUCCGUGGGCUGGGCCGUGAUCACCGACGAGUACAAGGUGCCCUCCAAGAAGUUCAAGGUGCUGGGCAACACCGACCGGCACUCCAUCAAGAAGAACCUGAUCGGCGCCCUGCUGUUCGACUCCGGCGAGACCGCCGAGGCCACCCGGCUGAAGCGGACCGCCCGGCGGCGGUACACCCGGCGGAAGAACCGGAUCUGCUACCUGCAGGAGAUCUUCUCCAACGAGAUGGCCAAGGUGGACGACUCCUUCUUCCACCGGCUGGAGGAGUCCUUCCUGGUGGAGGAGGACAAGAAGCACGAGCGGCACCCCAUCUUCGGCAACAUCGUGGACGAGGUGGCCUACCACGAGAAGUACCCCACCAUCUACCACCUGCGGAAGAAGCUGGUGGACUCCACCGACAAGGCCGACCUGCGGCUGAUCUACCUGGCCCUGGCCCACAUGAUCAAGUUCCGGGGCCACUUCCUGAUCGAGGGCGACCUGAACCCCGACAACUCCGACGUGGACAAGCUGUUCAUCCAGCUGGUGCAGACCUACAACCAGCUGUUCGAGGAGAACCCCAUCAACGCCUCCGGCGUGGACGCCAAGGCCAUCCUGUCCGCCCGGCUGUCCAAGUCCCGGCGGCUGGAGAACCUGAUCGCCCAGCUGCCCGGCGAGAAGAAGAACGGCCUGUUCGGCAACCUGAUCGCCCUGUCCCUGGGCCUGACCCCCAACUUCAAGUCCAACUUCGACCUGGCCGAGGACGCCAAGCUGCAGCUGUCCAAGGACACCUACGACGACGACCUGGACAACCUGCUGGCCCAGAUCGGCGACCAGUACGCCGACCUGUUCCUGGCCGCCAAGAACCUGUCCGACGCCAUCCUGCUGUCCGACAUCCUGCGGGUGAACACCGAGAUCACCAAGGCCCCCCUGUCCGCCUCCAUGAUCAAGCGGUACGACGAGCACCACCAGGACCUGACCCUGCUGAAGGCCCUGGUGCGGCAGCAGCUGCCCGAGAAGUACAAGGAGAUCUUCUUCGACCAGUCCAAGAACGGCUACGCCGGCUACAUCGACGGCGGCGCCUCCCAGGAGGAGUUCUACAAGUUCAUCAAGCCCAUCCUGGAGAAGAUGGACGGCACCGAGGAGCUGCUGGUGAAGCUGAACCGGGAGGACCUGCUGCGGAAGCAGCGGACCUUCGACAACGGCUCCAUCCCCCACCAGAUCCACCUGGGCGAGCUGCACGCCAUCCUGCGGCGGCAGGAGGACUUCUACCCCUUCCUGAAGGACAACCGGGAGAAGAUCGAGAAGAUCCUGACCUUCCGGAUCCCCUACUACGUGGGCCCCCUGGCCCGGGGCAACUCCCGGUUCGCCUGGAUGACCCGGAAGUCCGAGGAGACCAUCACCCCCUGGAACUUCGAGGAGGUGGUGGACAAGGGCGCCUCCGCCCAGUCCUUCAUCGAGCGGAUGACCAACUUCGACAAGAACCUGCCCAACGAGAAGGUGCUGCCCAAGCACUCCCUGCUGUACGAGUACUUCACCGUGUACAACGAGCUGACCAAGGUGAAGUACGUGACCGAGGGCAUGCGGAAGCCCGCCUUCCUGUCCGGCGAGCAGAAGAAGGCCAUCGUGGACCUGCUGUUCAAGACCAACCGGAAGGUGACCGUGAAGCAGCUGAAGGAGGACUACUUCAAGAAGAUCGAGUGCUUCGACUCCGUGGAGAUCUCCGGCGUGGAGGACCGGUUCAACGCCUCCCUGGGCACCUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAGGAGAACGAGGACAUCCUGGAGGACAUCGUGCUGACCCUGACCCUGUUCGAGGACCGGGAGAUGAUCGAGGAGCGGCUGAAGACCUACGCCCACCUGUUCGACGACAAGGUGAUGAAGCAGCUGAAGCGGCGGCGGUACACCGGCUGGGGCCGGCUGUCCCGGAAGCUGAUCAACGGCAUCCGGGACAAGCAGUCCGGCAAGACCAUCCUGGACUUCCUGAAGUCCGACGGCUUCGCCAACCGGAACUUCAUGCAGCUGAUCCACGACGACUCCCUGACCUUCAAGGAGGACAUCCAGAAGGCCCAGGUGUCCGGCCAGGGCGACUCCCUGCACGAGCACAUCGCCAACCUGGCCGGCUCCCCCGCCAUCAAGAAGGGCAUCCUGCAGACCGUGAAGGUGGUGGACGAGCUGGUGAAGGUGAUGGGCCGGCACAAGCCCGAGAACAUCGUGAUCGAGAUGGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACUCCCGGGAGCGGAUGAAGCGGAUCGAGGAGGGCAUCAAGGAGCUGGGCUCCCAGAUCCUGAAGGAGCACCCCGUGGAGAACACCCAGCUGCAGAACGAGAAGCUGUACCUGUACUACCUGCAGAACGGCCGGGACAUGUACGUGGACCAGGAGCUGGACAUCAACCGGCUGUCCGACUACGACGUGGACCACAUCGUGCCCCAGUCCUUCCUGAAGGACGACUCCAUCGACAACAAGGUGCUGACCCGGUCCGACAAGAACCGGGGCAAGUCCGACAACGUGCCCUCCGAGGAGGUGGUGAAGAAGAUGAAGAACUACUGGCGGCAGCUGCUGAACGCCAAGCUGAUCACCCAGCGGAAGUUCGACAACCUGACCAAGGCCGAGCGGGGCGGCCUGUCCGAGCUGGACAAGGCCGGCUUCAUCAAGCGGCAGCUGGUGGAGACCCGGCAGAUCACCAAGCACGUGGCCCAGAUCCUGGACUCCCGGAUGAACACCAAGUACGACGAGAACGACAAGCUGAUCCGGGAGGUGAAGGUGAUCACCCUGAAGUCCAAGCUGGUGUCCGACUUCCGGAAGGACUUCCAGUUCUACAAGGUGCGGGAGAUCAACAACUACCACCACGCCCACGACGCCUACCUGAACGCCGUGGUGGGCACCGCCCUGAUCAAGAAGUACCCCAAGCUGGAGUCCGAGUUCGUGUACGGCGACUACAAGGUGUACGACGUGCGGAAGAUGAUCGCCAAGUCCGAGCAGGAGAUCGGCAAGGCCACCGCCAAGUACUUCUUCUACUCCAACAUCAUGAACUUCUUCAAGACCGAGAUCACCCUGGCCAACGGCGAGAUCCGGAAGCGGCCCCUGAUCGAGACCAACGGCGAGACCGGCGAGAUCGUGUGGGACAAGGGCCGGGACUUCGCCACCGUGCGGAAGGUGCUGUCCAUGCCCCAGGUGAACAUCGUGAAGAAGACCGAGGUGCAGACCGGCGGCUUCUCCAAGGAGUCCAUCCUGCCCAAGCGGAACUCCGACAAGCUGAUCGCCCGGAAGAAGGACUGGGACCCCAAGAAGUACGGCGGCUUCGACUCCCCCACCGUGGCCUACUCCGUGCUGGUGGUGGCCAAGGUGGAGAAGGGCAAGUCCAAGAAGCUGAAGUCCGUGAAGGAGCUGCUGGGCAUCACCAUCAUGGAGCGGUCCUCCUUCGAGAAGAACCCCAUCGACUUCCUGGAGGCCAAGGGCUACAAGGAGGUGAAGAAGGACCUGAUCAUCAAGCUGCCCAAGUACUCCCUGUUCGAGCUGGAGAACGGCCGGAAGCGGAUGCUGGCCUCCGCCGGCGAGCUGCAGAAGGGCAACGAGCUGGCCCUGCCCUCCAAGUACGUGAACUUCCUGUACCUGGCCUCCCACUACGAGAAGCUGAAGGGCUCCCCCGAGGACAACGAGCAGAAGCAGCUGUUCGUGGAGCAGCACAAGCACUACCUGGACGAGAUCAUCGAGCAGAUCUCCGAGUUCUCCAAGCGGGUGAUCCUGGCCGACGCCAACCUGGACAAGGUGCUGUCCGCCUACAACAAGCACCGGGACAAGCCCAUCCGGGAGCAGGCCGAGAACAUCAUCCACCUGUUCACCCUGACCAACCUGGGCGCCCCCGCCGCCUUCAAGUACUUCGACACCACCAUCGACCGGAAGCGGUACACCUCCACCAAGGAGGUGCUGGACGCCACCCUGAUCCACCAGUCCAUCACCGGCCUGUACGAGACCCGGAUCGACCUGUCCCAGCUGGGCGGCGACGGCGGCGGCUCCCCCAAGAAGAAGCGGAAGGUGUGACUAGCACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAUUCUCUCGAG

indicates data missing or illegible when filed

1.2 Human KLKB1 Guide Design and Human KLKB1 with Cynomolgus HomologyGuide Design

Guide RNAs were designed toward human KLKB1 (ENSG00000164344) targetingthe protein coding regions within Exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, and 15. Guide RNAs were also designed toward cynomolgusKLKB1 (ENSMFAT00000002355) targeting the protein coding regions withinExons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. Guide RNAsand corresponding target genomic coordinates are provided above (Table1).

1.2. Cas9 (mRNA/Protein) and Guide RNA Delivery In Vitro

1.2.1. Cell Preparation, Delivery In Vitro

Primary human hepatocytes (PHH) (Gibco, Lots Hu8296, Hu8300, and Hu8284,Hu8296, HU8290, and HU8317) and primary cynomolgus hepatocytes (PCH)(Gibco, Lots Cy367, Cy400, and 10281011) were thawed and resuspended inhepatocyte thawing medium with supplements (Gibco, Cat. CM7500) followedby centrifugation. The supernatant was discarded and the pelleted cellsresuspended in hepatocyte plating medium (William's E Medium(Invitrogen, Cat. A1217601) with Dexamethosone+cocktail supplement, FBScontent, and Plating Supplements (Gibco, Cat. CM3000)). Cells werecounted and plated on Bio-coat collagen I coated 96-well plates(ThermoFisher, Cat. 877272) at a density of 30,000-35,000 cells/well forPHH and 40,000-45,000 cells/well for PCH. Plated cells were allowed tosettle and adhere for 4-6 hours in a tissue culture incubator at 37° C.and 5% CO₂ atmosphere. After incubation cells were checked for monolayerformation and were washed once with hepatocyte maintenance medium(William's E Medium with maintenance supplements (Gibco, Cat. CM4000))or Cellartis Power Primary HEP Medium (Takada, Cat. Y20020).

Guide RNAs targeting KLKB1 were delivered to cells using a liposomalsystem with Cas9 protein, for example, or using an LNP formulationcomprising Cas9 mRNA and guide RNA as further described below.

1.2.2. RNP Transfection

RNP transfection was used with a liposomal system (Lipofectamine RNAiMAX(ThermoFisher, Cat. 13778150) and CRISPR reagents (guide RNA, Cas9Protein) to shuttle a ribonucleoprotein (RNP) complex across the cellmembrane.

For studies utilizing dual guide (dgRNA), individual crRNA and trRNA waspre-annealed by mixing equivalent amounts of reagent and incubating at95° C. for 2 min and cooling to room temperature. The gRNA consisting ofpre-annealed crRNA and trRNA was added to Spy Cas9 protein in thereaction buffer (OptiMem) to form a RNP complex and the formed RNPcomplex was incubated at room temperature for 10 minutes. The RNPcomplex was diluted with OptiMem to prepare a 1 μm RNP complex stocksolution. A Transfection Mix including Lipofectamine RNAiMAX and OptiMemwas prepared and incubated for at least 5 minutes. The Transfection Mixwas added to the RNP complex and incubated at room temperature for 10minutes, and the Transfection Agent (Transfection Mix and RNP complex)was added to cells. Cells were transfected with the RNP complexcontaining Spy Cas9 protein (10 nM), individual guide/tracer RNA (10nM), and Lipofectamine RNAiMAX (1.0 μL/well) and OptiMem.

1.2.1 RNP Electroporation

RNP Electroporation was used with the cell electroporation system (Lonza4D Nucleofector™ kit 816B0346) and CRISPR reagents, gRNA and Cas9protein, to shuttle a ribonucleoprotein (RNP) complex across the cellmembrane.

For studies utilizing dgRNA, individual crRNA and trRNA werepre-annealed by mixing equivalent amounts of reagent and incubating at95° C. for 2 min and cooling to room temperature.

For studies utilizing sgRNA, a 50 uM sgRNA stock solution was preparedby incubating equal amounts of 100 uM sgRNA to water at 95° C. for 2 minfollowed by cooling on ice for 5 minutes. The sgRNA was added to the SpyCas9 protein in reaction buffer (20 mM Hepes, 100 mM KCl, 1 mM MgCl2,10% glycerol, 1 mM DTT pH 7.5) to form an RNP complex and incubated atroom temperature for 10 minutes. Cells were electroporated (Amaxa™96-well Shuttle™ Cat. AAM-1001S) with the RNP complex containing SpyCas9 protein (2 uM) and gRNA (4 uM) and Lonza P3 buffer (Catalog #:V4SP-3960). Post electroporation, hepatocyte plating media (Will's E,Cat. A12176-01) was added to the cell plate, the media with cells wastransferred to collagen coated plates (Corning 354407). After 4-6 hrs,the media was changed to maintenance media (William's E (Gibco, Cat.A12176-01, Lot 2039733) and maintenance supplement (Gibco, Cat.A12176-01, Lot 2039733) for overnight incubation at 37° C. overnight.

1.2.4. Preparation of LNP Formulation Containing sgRNA and Cas9 mRNA

In general, the lipid nanoparticle components were dissolved in 100%ethanol at various molar ratios. The RNA cargos (e.g., Cas9 mRNA andsgRNA) were dissolved in 25 mM citrate, 100 mM NaCl, pH 5.0, resultingin a concentration of RNA cargo of approximately 0.45 mg/mL. The LNPsused in Examples 2-10 contained ionizable lipid((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate, also called3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate), cholesterol, DSPC, and PEG2k-DMG in a50:38:9:3 molar ratio, respectively. The LNPs were formulated with alipid amine to RNA phosphate (N:P) molar ratio of about 6, and a ratioof gRNA to mRNA of 1:2 by weight. The LNPs used in Examples 2-10comprise a Cas9 mRNA and an sgRNA.

The LNPs were prepared using a cross-flow technique utilizing impingingjet mixing of the lipid in ethanol with two volumes of RNA solutions andone volume of water. The lipid in ethanol was mixed through a mixingcross with the two volumes of RNA solution. A fourth stream of water wasmixed with the outlet stream of the cross through an inline tee (SeeWO2016010840 FIG. 2 ). The LNPs were held for 1 hour at roomtemperature, and further diluted with water (approximately 1:1 v/v).Diluted LNPs were concentrated using tangential flow filtration on aflat sheet cartridge (Sartorius, 100 kD MWCO) and then buffer exchangedusing PD-10 desalting columns (GE) into 50 mM Tris, 45 mM NaCl, 5% (w/v)sucrose, pH 7.5 (TSS). The resulting mixture was then filtered using a0.2 μm sterile filter. The final LNPs were characterized to determinethe encapsulation efficiency, polydispersity index, and average particlesize. The final LNP was stored at 4° C. or −80° C. until further use.

1.2.5. sgRNA and Cas9 mRNA Lipofection

Lipofection of Cas9 mRNA and gRNAs used pre-mixed lipid formulations.The lipofection reagent contained ionizable lipid((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate, also called3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate), cholesterol, DSPC, and PEG2k-DMG in a50:38:9:3 molar ratio, respectively. This mixture was reconstituted in100% ethanol then mixed with RNA cargos (e.g., Cas9 mRNA and gRNA) at alipid amine to RNA phosphate (N:P) molar ratio of about 6.0. Guide RNAwas chemically synthesized by commercial vendors or using standard invitro synthesis techniques with modified nucleotides. A Cas9 ORF ofTable 5 was produced by IVT as described in WO2019/067910, see e.g.paragraph [00354], using a 2 hour IVT reaction time and purifying themRNA by LiCl precipitation followed by tangential flow filtration.Lipofections were performed with 3% cynomolgus serum and a ratio of gRNAto mRNA of 1:1 by weight.

1.2.6. LNP Transfection

Modified sgRNAs targeting human KLKB1 were formulated in LNPs asdescribed in Example 1. Primary human hepatocytes were plated asdescribed in Example 1. Cells were incubated at 37° C., 5% CO2 for 48hours prior to treatment with LNPs. LNPs were incubated in mediacontaining 3% fetal bovine serum (FBS) at 37° C. for 10 minutes.Post-incubation, media was aspirated from cells and the mixture of mediawith 3% FBS and LNP were added to the hepatocytes. At 72 to 96 hourspost-transfection, a portion of the cells were collected and processedfor NGS sequencing as described in Example 1.

1.3. Genomic DNA Isolation

Transfected PHH and PCH were harvested at 48 or 72 hourspost-transfection. The gDNA was extracted from each well of a 96-wellplate using 50 μL/well BuccalAmp DNA Extraction solution (Epicentre,Cat. QE09050) or Zymo's Quick RNA/DNA extraction kit (Cat. R2130)according to manufacturer's protocol. All DNA samples were subjected toPCR and subsequent NGS analysis, as described herein.

1.4. Next-Generation Sequencing (“NGS”) and Analysis for On-TargetCleavage Efficiency

To quantitatively determine the efficiency of editing at the targetlocation in the genome, next generation sequencing was utilized toidentify the presence of insertions and deletions introduced by geneediting. PCR primers were designed around the target site within thegene of interest (e.g. KLKB1), and the genomic area of interest wasamplified. Primer sequence design was done as is standard in the field.

Additional PCR was performed according to the manufacturer's protocols(Illumina) to add chemistry for sequencing. The amplicons were sequencedon an Illumina MiSeq instrument. The reads were aligned to the human(e.g., hg38) reference genome after eliminating those having low qualityscores. The resulting files containing the reads were mapped to thereference genome (BAM files), where reads that overlapped the targetregion of interest were selected and the number of wild-type readsversus the number of reads which contain an insertion or deletion(“indel”) was calculated.

The editing percentage (e.g., the “editing efficiency” or “percentediting”) is defined as the total number of sequence reads withinsertions or deletions (“indels”) over the total number of sequencereads, including wild type.

A biochemical assay (See, e.g., Cameron et al., Nature Methods. 6,600-606; 2017) was used to discover potential off-target genomic sitescleaved by Cas9 targeting KLKB1. Purified genomic DNA (gDNA) from cellswere digested with in vitro assembled ribonucleoprotein (RNP) of Cas9and sgRNA, to induce DNA cleavage at the on-target site and potentialoff-target sites with homology to the sgRNA spacer sequence. After gDNAdigestion, the free gDNA fragment ends were ligated with adapters tofacilitate edited fragment enrichment and NGS library construction. TheNGS libraries were sequenced and through bioinformatic analysis, thereads were analyzed to determine the genomic coordinates of the free DNAends. Locations in the human genome with an accumulation of reads werethen annotated as potential off-target sites.

In known off-target detection assays, such as the biochemical assay usedabove, a large number of potential off-target sites are typicallyrecovered, by design, so as to “cast a wide net” for potential sitesthat can be validated in other contexts, e.g., in a primary cell ofinterest. For example, the biochemical assay typically overrepresentsthe number of potential off-target sites as the assay utilizes purifiedhigh molecular weight genomic DNA free of the cell environment and isdependent on the dose of Cas9 RNP used. Accordingly, potentialoff-target sites identified by these assays were validated usingtargeted sequencing of the identified potential off-target sites.

In one approach to targeted sequencing, Cas9 and a sgRNA of interest(e.g., a sgRNA having potential off-target sites for evaluation) wereintroduced to PHH or PCH cells. The cells were then lysed and primersflanking the potential off-target site(s) were used to generate anamplicon for NGS analysis. Identification of indels at a certain levelcan be used to validate potential off-target site, whereas the lack ofindels found at the potential off-target site can indicate a falsepositive in the off-target assay that was utilized.

Guides showing on target indel activity were tested for potentialoff-target genomic cleavage sites with this assay. Repair structureswere manually inspected at loci with statistically relevant indel ratesat the off-target cleavage sites to validate the repair structures.

1.5 Transcript Analysis by Quantitative PCR

Quantitative PCR was performed to assess KLKB1 transcript levels. Toisolate mRNA, the Qiagen RNeasy Mini Kit (Qiagen, Cat. 74106) was used.The RNeasy Mini Kit procedure was completed according to themanufacturer's protocol.

RNA was quantified using a Nanodrop 8000 (ThermoFisher Scientific, Cat.ND-8000-GL). The RNA quantification procedure was completed according tothe manufacturer's protocol. RNA samples were stored at −20° C. prior touse.

The Taqman RNA-to-Ct 1-Step Kit (Thermo Fisher Scientific, Cat. 4392938)was used to create the PCR reactions. The reaction set-up was completedaccording to the manufacturer's protocol. Alternatively, a Cells-to-CT1-Step TaqMan Kit (Thermo Fisher Scientific, Cat. A25603) was used toproduce samples for qPCR. Quantitative PCR probes targeting human orcynomolgus monkey KLKB1 (Thermo Fisher Scientific, Cat. 4351372,transcript UniGene ID Hs01111828_m1; Thermo Fisher Scientific, Cat.4331182, transcript UniGene ID Hs00168478_ml), internal control PPIB(Thermo Fisher Scientific, Cat. 4351372, transcript UniGene IDHs00168719_ml; Thermo Fisher Scientific, Cat. 4331182, transcriptUniGene ID Mf02802985 ml), internal control GAPDH (Thermo FisherScientific, Cat. 4351372, transcript UniGene ID Hs02786624_g1), andinternal control 18S (Thermo Fisher Scientific, Cat. 4319413E) were usedin the PCR reactions. The StepOnePlus Real-Time PCR System (ThermoFisher Scientific, Cat. 4376600) was used to perform the real-time PCRreaction and transcript quantification according to the manufacturer'sprotocol.

Double delta Ct analysis of KLKB1 mRNA was provided using the Ct valuesdetermined from the StepOnePlus Real-Time PCR System. A double delta Ctvalue was calculated for the Ct values for internal controls within eachsample compared to the values for KLKB1. The expression fold change wasdetermined based on the double delta Ct value for each sequence.

1.6. Protein Analysis of Tissue Culture Media by ELISA

PHH or PCH were transfected as previously described. Starting at threedays post-transfection and plating (96-well plate), media was changed oncells every two days. Seven to ten days post-transfection, media wasremoved from cells and then replaced with 100 μL of William's E culturemedia or Cellartis Power Primary HEP Medium (Takada, Cat. Y20020).Twenty-four to forty-eight hours later, media was harvested and storedat −20° C. Total secreted KLKB1 protein levels were determined using aprekallikrein ELISA Kit (Abcam, Cat. ab202405), which detectsprekallikrein and kallikrein (also, called total kallikrein). Kitreagents and standards were prepared using the manufacturer's protocols.Prior to running the ELISA, frozen media was thawed at room temperatureand centrifuged at 1000 rpm for 1 minute to pellet debris and thenplaced on ice. For the ELISA, 10 to 40 μL of media was diluted withSample Diluent NS assay diluent to a total volume of 50 ul. The ELISAprocedure was completed according to the manufacturer's protocol. Theplate was read on a SpectraMax M5 plate reader. Total kallikrein levelswere calculated by SoftMax Pro software ver. 6.4.2 using afour-parameter logistic curve fit of the standard curve. Reduction oftotal secreted pre-kallikrein protein for cells treated with KLKB1reagents was determined when compared to wells treated with controlreagents or untreated samples.

1.6.1 Protein Analysis of Serum by ELISA

Serum pre-kallikrein levels were measured in humanized mice using thefollowing procedure. Six to seven days post-dose, animals wereeuthanized by exsanguination via cardiac puncture under isoflouraneanesthesia. Blood was collected into serum separator tubes, and allowedto clot at room temperature for 2 hours before being spun down at 9000 gfor 10 min to separate the serum. Samples were stored at −20 C untilanalysis.

Pre-kallikrein protein levels were determined using a humanpre-kallikrein ELISA Kit (Abcam, Cat. ab202405), which detectsprekallikrein and kallikrein (also, called total kallikrein). Briefly,sera were serial diluted with kit sample diluent to a final dilution of1:500 or 1:1000 fold before adding to the ELISA plate. The assay wascarried out according to the manufacturer's protocol. The plate was readon a Clariostar plate reader (BMG Labtech). Pre-kallikrein levels werecalculated by Mars software using a four-parameter logistic curve fit ofthe standard curve. Reduction of total secreted pre-kallikrein proteinfor cells treated with KLKB1 reagents was determined when compared towells treated with control reagents or untreated samples.

1.6.2. Protein Analysis by Western Blot

PHH were treated with LNP formulated with select guide RNAs from Table 1as further described below. LNPs were incubated in Cellartis PowerPrimary HEP Medium (Takada, Cat. Y20020) containing 3% FBS or cynomolgusserum at 37° C. for 10 minutes. Post-incubation the LNPs were added tothe human hepatocytes. Starting at three days post-transfection, mediawas changed on cells every two days. Ten to fourteen dayspost-transfection, for cells plated in a 96-well plate the media wasremoved and the cells were lysed with 50 μL/well RIPA buffer (Boston BioProducts, Cat. BP-115) plus freshly added protease inhibitor mixtureconsisting of complete protease inhibitor cocktail (Sigma, Cat.11697498001), 1 mM DTT, and 250 U/ml Benzonase (EMD Millipore, Cat.71206-3) per 30,000 to 45,000 cells. Cells were kept on ice for 30minutes at which time NaCl (1 M final concentration) was added. Celllysates were thoroughly mixed and retained on ice for 30 minutes. Thewhole cell extracts (“WCE”) were transferred to a PCR plate andcentrifuged to pellet debris. A Bradford assay (Bio-Rad, Cat. 500-0001)was used to assess protein content of the lysates. The Bradford assayprocedure was completed according to the manufacturer's protocol.Extracts were stored at −20° C. prior to use.

Western blots were performed to assess KLKB1 protein levels. Lysateswere mixed with Laemmli buffer (Boston BioProducts, Cat. BP-111R) anddenatured at 95° C. for 10 minutes. Western blots were run using theNuPage system on 4-12% Bis-Tris gels (Thermo Fisher Scientific, Cat.NP0323BOX) according to the manufacturer's protocol followed by wettransfer onto 0.45 μm nitrocellulose membrane (Bio-Rad, Cat. 1620115).After transfer membranes were rinsed thoroughly with water and stainedwith Ponceau S solution (Boston Bio Products, Cat. ST-180) to confirmcomplete and even transfer. Blots were blocked using 5% dry milk in TBSfor 30 minutes on a lab rocker at room temperature. Blots were rinsedwith TBST and probed with rabbit α-kallikrein monoclonal antibody(Abcam, Cat. ab124938) at 1:1000 in TBST. For blots with in vitro celllysate, GAPDH was used as a loading control (Novus, Cat. NB600502) at1:2500 in TBST and incubated simultaneously with the KLKB1 primaryantibody. After incubation, blots were rinsed 3 times for 5 minutes eachin TBST. Blots were visualized and analyzed by densitometry using aLicor Odyssey system.

1.6.3. Electrochemiluminescence-Based Detection of Plasma KallikreinLevels

Plasma kallikrein levels in in samples were measured by an immunoassayusing an electrochemiluminescence detection platform by MesoScaleDiscovery (MSD). A 96-well MSD standard plate (Cat. No: L15XA) wascoated with 25 or 40 μL of a mouse monoclonal capture antibody forkallikrein (LS-Bio, LS-C38308) at a concentration of 1 μg/mL in PBS overnight at 4° C. On the following day, the wells were washed and thenblocked with 150 μL of 3% Blocker-A (MSD, Cat. No: R93AA) and incubatedfor 1 hour at room temperature on a shaker set to 700 rpm. Afterwashing, the samples for the determination of kallikrein concentrationalong with a human kallikrein standard of a known concentration, madein-house, were added to the wells and incubated for 2 hours at roomtemperature on a shaker set to 700 rpm. Both samples and standards werediluted in 1% Blocker-A, optionally with 0.05% Tween20.

After washing, a 25 μL of the detection antibody solution was added(LSBio #C185168 at 1 ug/mL and MSD #R32AG at 500 ug/mL in 1% Blocker-Awith 0.05% Tween20) and incubated for 1 hour at room temperature. Theplate was washed and 150 μL MSD gold Read Buffer (MesoScale Discovery,Cat. No: R92TG) was added to each well. The plate was read using theQuickPlex SQ 120 (MesoScale Discovery). The plate was washed 3× with PBSwith 0.05% Tween20 between the different steps.

1.7. Fluorometric analysis of plasma kallikrein activity

Total plasma kallikrein activity levels in samples, such as non-humanprimate (NHP) samples, were measured using the Fluorometric SensoLyteRh110 Plasma Kallikrein Activity Assay Kit (Anaspec Cat No: AS-72255). Achloroform pretreatment was performed to inhibit C1-Inhibitor activityby mixing an equal volume of cold chloroform with K2EDTA NHP plasma in a96-well plate. The plate was then centrifuged for 5 min at 4° C. at16,000× g and 10 uL of the treated plasma was carefully collected fromthe top layer. In a 96-well black microplate, the 10 uL of pretreatedplasma was mixed with 30 uL of assay buffer, 10 uL of PlasmaPrekallikrein Activator and 50 uL of substrate, all of which areprovided in the kit and prepared according to kit protocol. Thefluorescence measurements were immediately initiated at Ex/Em=490 nm/520nm with a reading every 5 minutes for 1 hour on a SpectraMax M5 platereader. The slope of the linear portion of the kinetic fluorometricreadout for a given post-treatment plasma sample is compared to theslope of the pre-treatment plasma sample from the same animal tocalculate % basal.

1.7.1 Electrochemiluminescence-Based Detection of Plasma KallikreinLevels in Non-Human Primates

Plasma kallikrein levels in non-human primates (NHP) were measured by animmunoassay using an electrochemiluminescence detection platform byMesoScale Discovery (MSD). A 96-well MSD standard plate (Cat. No: L15XA)was coated with 40 μL of a mouse monoclonal capture antibody forkallikrein (LS-Bio, LS-C38308) at a concentration of 1 pg/mL in PBS overnight at 4° C. On the following day, the wells were washed and thenblocked with 150 μL of 3% Blocker-A (MSD, Cat. No: R93AA) and incubatedfor 1 hour at room temperature on a shaker set to 700 rpm. Afterwashing, NHP samples for the determination of kallikrein concentrationalong with a NHP kallikrein standard of a known concentration, madein-house, were added to the wells and incubated for 2 hours at roomtemperature on a shaker set to 700 rpm. Both samples and standards werediluted in 1% Blocker-A with 0.05% Tween20.

After washing, 25 μL of the detection antibody solution was added andincubated for 1 hour at room temperature. The plate was washed and 150μL MSD gold Read Buffer (MesoScale Discovery, Cat. No: R92TG) was addedto each well. The plate was read using the QuickPlex SQ 120 (MesoScaleDiscovery). The plate was washed 3× with PBS with 0.05% Tween 20 betweenthe different steps.

1.8 Vascular Permeability Assay

The Evans Blue vascular permeability assay is an established model ofedema and vascular leakage that can be used as a model in the study ofHAE (see, e.g., Bhattacharjee et al., 2013). The assay is based on theinjection of Evans Blue, an albumin-binding dye, in a test animal,typically a mouse. Under physiologic conditions the endothelium isimpermeable to albumin, so the albumin-bound Evans blue remainsrestricted within blood vessels. In pathologic conditions that promoteincreased vascular permeability, extravasation of Evans Blue can bereadily observed qualitatively e.g., by the presence of blue color inthe ears, feet, and nose of mice after intravenous injection, orquantitatively by measurement of dye incorporated into tissue, e.g.,intestine.

Using the huKLKB1 mouse, a model for vascular permeability was developedto evaluate the potential of KLKB1 editing to mitigate the effects ofexcess bradykinin production (Bhattacharjee et al., 2013). A modifiedKLKB1 targeting sgRNA and the Cas9 mRNA was administered in a doseresponse at total RNA doses of 0.03 mg/kg, 0.1 mg/kg and 0.3 mg/kg.Additional groups were treated with 0.3 mg/kg of non-targeting-LNPcontrol and TSS vehicle control. Thirteen days post-dose, vascularpermeability was induced using a 2.5 mg/kg intraperitoneal injection ofthe angiotensin converting enzyme (ACE) inhibitor captopril. After 15minutes, a mixture of Evans Blue Dye (30 mg/kg) and dextran sulfate (0.3mg/kg) was administered by intravenous tail injection. The animals wereeuthanized 15 minutes after this injection and evaluated for dyeextravasation into the colon by optical density (OD) at the absorbanceof 600 nm via the Clariostar plate reader (BMG LabTech). Liver and serumwere collected to quantify huKLKB1 gene editing and kallikrein protein,respectively.

Example 2: Screening and In Vitro Guide Characterization

2.1. Screening of Dual Guide RNAs (dgRNAs) that Target Human KLKB1

Guides targeting human KLKB1 were prepared as dual guide RNAs andevaluated by transfection into primary human hepatocytes (PHH) andprimary cynomolgus hepatocytes (PCH) as described in Example 1. Thecells were lysed 48 hours post treatment for NGS analysis as describedin Example 1. The guides shown in Table 6 were tested.

TABLE 6 Dual guides and single guides in human and cynomolgus SEQ humanID human cyno dgRNA human guide sequence NO sgRNA sgRNA CR005916ACAGGAAACUGUAGCAAACA   1 G012253 NA CR005917 AUAGAUAAUUCACUUACCAC   2G012254 NA CR005918 UACAUCCCCACCUCUGAAGA   3 G012255 NA CR005919AACUGAAUAGCAAACACCUU  89 NA NA CR005920 ACAAUUACCAAUUUCUGAAA  90 NA NACR005921 UACAAUUACCAAUUUCUGAA  91 NA NA CR005922 UCUUGAGGAGUAGAGGAACU  4 G012256 NA CR005923 GGUGUUUUCUUGAGGAGUAG  92 NA NA CR005924ACCAGGUAAAGUUCUUUUGC   5 G012257 NA CR005925 GGGUAAAUUUUAGAAUGGCA   6G012258 NA CR005926 CGGGUAAAUUUUAGAAUGGC  93 NA G013884 CR005927CUCCCGGGUAAAUUUUAGAA  94 NA G013925 CR005928 AUUUACCCGGGAGUUGACUU   7G012259 NA CR005929 UACCCGGGAGUUGACUUUGG   8 G012260 NA CR005930UAUGGGACACAAGGGAGCUC  95 NA NA CR005931 UCUUUGAGAUUGUGUAACAC   9 G012261NA CR005932 CUUUGAGAUUGUGUAACACU  10 G012262 NA CR005933UUUGAGAUUGUGUAACACUG  11 G012263 NA CR005934 UUGGAGGAACAAACUCUUCU  96 NAG013912 CR005935 UGGAGGAACAAACUCUUCUU  97 NA NA CR005936CAAACUCUUCUUGGGGAGAG  98 NA NA CR005937 CUAUGAGUGACCCUCCACAC  99 NAG013886 CR005938 CUGUGUGGAGGGUCACUCAU 100 NA G013938 CR005939GGUCACUCAUAGGACACCAG 101 NA G013946 CR005940 GUCACUCAUAGGACACCAGU 102 NAG013896 CR005941 ACUGCUGCCCACUGCUUUGA 103 NA NA CR005942ACACUUACCCAUCAAAGCAG 104 NA G013902 CR005943 UACAUACCAGUGUAAUUCAA  12G012264 NA CR005944 AGGAACACCUACCGCUAUAA 105 NA G013871 CR005945CUCCGGGACUGUACUUUAAU 106 NA G013889 CR005946 GUCCCAUACGCAAUCCUAGU 107 NAG013890 CR005947 CUCAGCACCUUUAUAGCGGU 108 NA G013892 CR005948UAUAGCGGUAGGUGUUCCUC 109 NA G013874 CR005949 CUCCAACUAGGAUUGCGUAU  13G012265 G013933 CR005950 CUAUUAAAGUACAGUCCCGG 110 NA G013875 CR005951AGGAUUGCGUAUGGGACACA  14 G012266 G013904 CR005952 GGAUUGCGUAUGGGACACAA 15 G012267 G013901 CR005953 GUGCUGAGUAACGUGGAAUC 111 NA G013883CR005954 UAUAAAGGUGCUGAGUAACG 112 NA G013878 CR005955UCUCCAACUAGGAUUGCGUA 113 NA G013908 CR005956 GUUACUCAGCACCUUUAUAG  16G012268 G013945 CR005957 AUAGCGGUAGGUGUUCCUCC 114 NA G013873 CR005958CUGCCAAAAGUACAUCGAAC 115 NA G013877 CR005959 UGCCUAUUAAAGUACAGUCC  17G012269 NA CR005960 CUAUGGAUGGUUCUCCAACU  18 G012270 G013922 CR005961ACCAAUUUCUGAAAGGGCAC 116 NA NA CR005962 GUGUUUCUUAAGAUUAUCUA 117 NA NACR005963 GAUGUUUGGCGCAUCUAUAG  19 G012271 G013921 CR005964CCAAUUUCUGAAAGGGCACA 118 NA NA CR005965 UUCUUAAGAUUAUCUAUGGA 119 NAG013940 CR005966 CUGUUCGAUGUACUUUUGGC 120 NA NA CR005967UGUUCGAUGUACUUUUGGCA 121 NA G013880 CR005968 GGUGGAAUGUGCACCUCAUC 122 NAG013939 CR005969 GUCCGACACACAAAAGCAUC 123 NA G013894 CR005970AUGCGCCAAACAUCCUGCAG  20 G012272 G013885 CR005971 AAACUGGCAGCGAAUGUUAC124 NA G013930 CR005972 UGCCACGCAAACAUUUCACA 125 NA NA CR005973GCACCUGUUCGAUGUACUUU 126 NA G013870 CR005974 AGAUGCGCCAAACAUCCUGC 127 NANA CR005975 GCACCUCAUCUGGCAGUAUU 128 NA NA CR005976 CAUCUGAGAACGCAAGAUGC129 NA G013934 CR005977 AUGCCCAAUACUGCCAGAUG 130 NA NA CR005978UGCACCUCAUCUGGCAGUAU 131 NA G013944 CR005979 CUCCUUUAUAAAUGUCUCGA  21G012273 G013905 CR005980 AUGUCAUUGAUUGAACUUGC 132 NA G013936 CR005981ACAAGCACACGCAUUGUUGG 133 NA G013893 CR005982 UGUUACUGGUGCACCUUUUU  22G012274 NA CR005983 GAUGCGCCAAACAUCCUGCA  23 G012275 G013876 CR005984UAUCGCCUUGAUAAAACUCC 134 NA G013926 CR005985 CCUCAAGAAAACACCAUAUC 135 NAG013906 CR005986 AAACGCCUUCUUCAGAGGUG 136 NA NA CR005987AAAACAAGCACACGCAUUGU 137 NA G013891 CR005988 CAUCGAACAGGUGCAGUUUC 138 NAG013879 CR005989 GGCUUCCCCUGCAGGAUGUU 139 NA G013881 CR005990UUGAUGACCACAUUGCUUCA 140 NA G013937 CR005991 AGGAGCCUGGAGUUUUAUCA 141 NANA CR005992 AUCUGGCAGUAUUGGGCAUU  24 G012276 G013915 CR005993UGCCAUCGAGACAUUUAUAA 142 NA G013899 CR005994 GCGUGGCAUAUGAAAAAAAC  25G012277 NA CR005995 UAUAAAGGAGUUGAUAUGAG  26 G012278 G013913 CR005996AGCAAGUUCAAUCAAUGACA 143 NA G013897 CR005997 GGACAUUCCUUGAAGCAAUG 144 NANA CR005998 ACACCUUGAAUUGUACUCAC  27 G012279 NA CR005999GUUGGGGUGAUAGGUGCAGA 145 NA NA CR006000 GAAAACGCCUUCUUCAGAGG 146 NA NACR006001 UAUGAAAACGCCUUCUUCAG 147 NA NA CR006002 CUCAGAUGUGGAUGUUGCCA148 NA NA CR006003 CUCUCCUAGGCUUCCCCUGC 149 NA NA

Editing was determined for dgRNAs in two separate sets of PHH and PCHpopulations. The screening data for the guide sequences are listed inTable 6 above. Table 7A and FIGS. 1A-1B show the percent editing for theKLKB1 targeting guides co-transfected with Spy Cas9 protein in primaryhuman hepatocytes (PHH) (N=2) and Table 7B and FIGS. 1C-1D for primarycynomolgus hepatocytes (PCH) (N=2).

The top performing guide RNAs and corresponding editing data from Set 2are marked with an asterisk (*) in Table 7A and 7B. When compared, thesets were determined to be highly correlated (Spearman R=0.985).

TABLE 7A KLKB1 editing data for dual guides delivered to primary humanhepatocytes: Primer sets 1 & 2 SET 1 SET 2 GUIDE Avg % Std Dev % GUIDEAvg % Std Dev % ID Edit Edit ID Edit Edit CR005916* 44.8 4.67 CR005916*46.8 1.7 CR005917* 45.75 1.77 CR005917* 49.9 4.38 CR005918 ND NDCR005918 ND ND CR005919 24.6 0.28 CR005919 28.9 4.81 CR005920 9.35 0.35CR005920 10.7 0.99 CR005921 6.15 1.06 CR005921 5.55 0.64 CR005922 ND NDCR005922 19.25 1.91 CR005923 19.25 4.74 CR005923 20.35 2.76 CR005924 6.10.71 CR005924 5.55 0.78 CR005925* 35.55 1.06 CR005925* 36.3 1.41CR005926 11.95 4.45 CR005926 11.2 2.83 CR005927 29.2 4.24 CR005927 30.42.97 CR005928 ND ND CR005928 33.45 4.03 CR005929 ND ND CR005929 51.350.07 CR005930* 32.45 0.21 CR005930* 32.6 8.06 CR005931* 37.85 5.02CR005931* 33.15 1.63 CR005932* 62.25 3.04 CR005932* 63.25 4.6 CR005933*70.05 1.91 CR005933* 62.45 2.47 CR005934 17 2.12 CR005934 16.5 0.71CR005935 26.25 0.35 CR005935 26.45 3.75 CR005936 4.55 0.64 CR005936 50.71 CR005937* 32.6 0.99 CR005937* 32.45 1.63 CR005938* 39.25 8.7CR005938* 36.85 5.87 CR005939 27 0.57 CR005939 24.85 3.46 CR005940 16.72.12 CR005940 17.15 1.91 CR005941 8.9 0.85 CR005941 8.95 1.48 CR00594219.8 0.71 CR005942 20.05 0.49 CR005943* 38.3 6.08 CR005943* 38.05 3.32CR005944 23.55 1.91 CR005944 22.05 4.17 CR005945 21.95 1.63 CR00594523.05 1.63 CR005946 29.35 2.47 CR005946 27.4 2.55 CR005947 12.4 2.97CR005947 12.55 1.63 CR005948 15.2 1.56 CR005948 14.5 0.85 CR005949 19.152.33 CR005949 19.5 0.99 CR005950 21.6 2.12 CR005950 19.2 1.7 CR005951*44.9 1.41 CR005951* 43.45 3.75 CR005952* 63.4 3.11 CR005952* 64.4 2.26CR005953 5.7 0 CR005953 6.35 1.06 CR005954 12.5 1.7 CR005954 12.8 0.57CR005955 24.65 3.04 CR005955 24.95 0.64 CR005956* 31.35 0.92 CR005956*30.55 4.88 CR005957 22.95 2.05 CR005957 24.8 1.41 CR005958 16.45 1.2CR005958 12.05 2.33 CR005959* 42.4 3.11 CR005959* 42.95 4.17 CR005960*38.7 0.57 CR005960* 41.1 4.38 CR005961 15.2 0.71 CR005961 13.8 3.25CR005962* 31.4 0 CR005962* 29.65 0.92 CR005963 ND ND CR005963 45.25 9.97CR005964 17.45 1.91 CR005964 15.65 3.89 CR005965 25.25 2.33 CR00596522.25 0.21 CR005966 8.35 2.9 CR005966 6.15 0.49 CR005967 19.5 2.55CR005967 16.7 3.11 CR005968 11.55 2.33 CR005968 11.65 1.91 CR005969 13.52.26 CR005969 12.35 3.75 CR005970 ND ND CR005970 22.25 4.17 CR005971 1.10.42 CR005971 1.15 0.64 CR005972 13.65 1.63 CR005972 12.1 2.97 CR0059736.45 0.21 CR005973 5.25 1.06 CR005974 ND ND CR005974 16.7 1.27 CR0059757.95 1.34 CR005975 6.75 0.92 CR005976 ND ND CR005976 14.6 0.99 CR00597712.2 2.69 CR005977 13.65 3.32 CR005978 9.95 1.06 CR005978 9.25 0.92CR005979 22.35 1.63 CR005979 22.15 0.21 CR005980 18.2 2.26 CR00598021.25 0.78 CR005981 18.2 1.27 CR005981 17.8 0.42 CR005982 6.25 1.77CR005982 3.95 2.76 CR005983* 53 NA CR005983* 43.3 9.9 CR005984 17.7 7.07CR005984 18.45 5.73 CR005985 15.1 8.91 CR005985 16.3 0.71 CR005986 ND NDCR005986 ND ND CR005987 10.6 2.12 CR005987 9.8 0.85 CR005988 8.55 2.05CR005988 6.4 1.13 CR005989 ND ND CR005989 4.9 0.42 CR005990 ND NDCR005990 20.2 6.36 CR005991 28.15 2.9 CR005991 26.9 4.81 CR005992 1.650.21 CR005992 4.85 0.92 CR005993* 38 1.27 CR005993* 31.35 1.2 CR005994*33.4 1.98 CR005994* 36.4 1.98 CR005995 22.55 2.47 CR005995 30.65 7.14CR005996 16.55 0.64 CR005996 13.75 3.46 CR005997 22.05 1.2 CR00599716.45 3.61 CR005998* 56.65 2.33 CR005998* 59.05 14.07 CR005999 25 3.68CR005999 22.45 2.19 CR006000 ND ND CR006000 ND ND CR006001 ND NDCR006001 ND ND CR006002 23.25 3.75 CR006002 20.85 2.05 CR006003 ND NDCR006003 7 1.13 *“selected dgRNA”, a subset of the tested guide RNAs

TABLE 7B KLKB1 editing data for crRNAs delivered to primary cynomolgushepatocytes: Sets 1 & 2 SET 1 SET 2 Avg Std Dev Avg Std Dev GUIDE ID %Edit % Edit GUIDE ID % Edit % Edit CR005916* 26.6 2.4 CR005916* 26.1 1.8CR005917* 31.5 3.3 CR005917* 33.2 2.8 CR005918* 0.6 0.2 CR005918 ND NDCR005919* 2.5 ND CR005919* 3.2 0.3 CR005920* 0.4 0.1 CR005920 0.1 0.1CR005921 0.1 0.0 CR005921 0.1 0.1 CR005010 72.8 0.2 CR005010 65.7 0.9CR005922* 0.5 0.1 CR005922* 0.6 0.3 CR005923 0.4 0.1 CR005923 0.3 0.1CR005924 0.2 0.0 CR005924 0.2 0.2 CR005925* 0.7 0.4 CR005925* 1.5 0.3CR005926 0.0 0.0 CR005926 0.0 0.0 CR005927* 3.1 0.8 CR005927* 3.2 0.2CR005928* 3.3 1.6 CR005928* 2.2 0.4 CR005929* 13.0 2.7 CR005929* 12.50.0 CR005930* 5.0 0.9 CR005930* 3.6 1.3 CR005931* 1.3 0.0 CR005931* 2.30.0 CR005932* 15.5 3.5 CR005932* 12.2 5.7 CR005933* 21.5 2.6 CR005933*16.6 ND CR005934 ND ND CR005934 0.3 0.2 CR005935 ND ND CR005935* 2.0 0.1CR005936 0.1 0.0 CR005936 ND ND CR005937* 2.8 0.1 CR005937* 2.9 0.1CR005938* 5.8 0.4 CR005938* 6.2 0.0 CR005939* 2.3 0.1 CR005939* 1.9 0.4CR005940* 1.2 0.3 CR005940* 1.2 0.3 CR005025 31.3 10.8 CR005025 36.5 NDCR005941 0.4 0.1 CR005941* 0.3 0.0 CR005942* 1.2 0.6 CR005942* 1.0 0.8CR005943* 4.0 0.4 CR005943* 3.1 0.0 CR005020 29.5 1.5 CR005020 30.7 0.6CR003187 14.5 0.6 CR003187 15.2 2.2 CR005964 0.3 0.4 CR005964 0.1 0.1CR005032 5.3 1.6 CR005032 4.5 0.9 *“selected dgRNA”, a subset of thetested guide RNAs

2.1.1 Cross Screening of sgRNAs in PHH and PCH, Editing

Selected guide sequences targeting KLKB1 were prepared as sgRNAs andfurther evaluated in PHH and PCH. PHH and PCH (Gibco, Lot Hu8298) wereprepared and transfected with RNP as described in Example 1. The cellswere lysed at 48 and 72 hours, respectively, post-treatment for NGSanalysis as described in Example 1. Table 8A and FIGS. 2A-2B showpercent editing in PHH and Table 8B and FIGS. 2C-2D in PCH.

TABLE 8A KLKB1 editing data for sgRNAs delivered to primary humanhepatocytes: Sets 1 & 2 SET 1 SET 2 Avg Std Dev Avg Std Dev GUIDE ID %Edit % Edit GUIDE ID % Edit % Edit G012321* 64.7 5.8 G012321* 62 5.5G012102 64 5.7 G012102 63.6 4.7 G012293* 63.5 4.4 G012293* 61.6 3.9G009246 63.3 3.1 G009246 62.3 4.6 G012308* 59.0 1.6 G012308* 59 1.5G012253* 58.6 3.5 G012253* 59.1 1.7 G012319* 55.3 0.9 G012319* 55.1 3.6G012298* 53.1 4.2 G012298* 54 4.9 G012320* 52.5 5.5 G012320* 51.9 7.7G012290* 52.0 1.4 G012290* 51.7 0.9 G012304* 48.7 2.7 G012304 NA NAG012323* 47.4 1.4 G012323* 49.1 1.1 G012280* 46.2 4.0 G012280* 45.4 5.4G012305* 46.1 5.2 G012305 NA NA G012303* 45.4 7.4 G012303 NA NA G012285*45.1 7.6 G012285* 45.2 8.6 G012335* 45.1 2.4 G012335* 44.7 3.2 G012286*44.7 5.3 G012286* 43.9 3.5 G000644 42.4 9.9 G000644 42.7 9.5 G012294*42.0 1.3 G012294* 43 2.4 G009267 41.6 0.6 G009267 45.8 1.3 G009285 37.71.7 G009285 35.6 1.3 G012334* 36.4 2.5 G012334* 36 5.3 G012325* 36.2 1.8G012325* 36.4 3.6 G012296* 35.9 6.2 G012296* 35.4 7.8 G012331 35.5 3.0G012331* 35.7 1.2 G012306 34.9 1.8 G012306 NA NA G012313 34.9 1.3G012313* 36.3 3.3 G012297 34.4 4.7 G012297 32.7 4.7 G012322 32.9 0.4G012322 33.2 0.4 G012299 32.4 0.7 G012299 NA NA G012333 31.3 0.4 G01233332.3 3.3 G012328 31.2 2.3 G012328* 34.9 0.1 G012309 30.4 0.1 G01230929.9 2.3 G009321 29.9 2 G009321 31.8 4.5 G012338 29.0 2.2 G012338 29.72.3 G012283 26.2 4.2 G012283 24.9 4.1 G012291 25.3 2.3 G012291 24.7 3.7G012302 23.2 NA G012302 24.6 3.1 G012337 23.0 2.3 G012337 23.6 0.5G012311 22.8 0.8 G012311 23.9 0.2 G012144 22.6 1.5 G012144 22.9 0.9G012327 22.4 3.5 G012327 21.7 3.4 G012316 22.3 0.1 G012316 21.1 0.5G000645 22 0.7 G000645 22.5 1.6 G012281 20.9 0.1 G012281 21.5 0.1G012300 19.2 2.6 G012300 NA NA G012310 19.2 2.5 G012310 18.2 0.1 G01232919.2 1.6 G012329 21.7 0.8 G012318 18.8 0.7 G012318 17.5 0.6 G012314 18.60.7 G012314 19.6 2.7 G012326 18.4 3.2 G012326 18.5 3.2 G012295 17.7 3.6G012295 17.6 5.7 G012287 17.2 0.4 G012287 17 2.4 G012288 16.7 0.1G012288 15.4 0.3 G012339 16.5 2.6 G012339 17.6 1.3 G012284 15.3 0.3G012284 15.4 1.5 G012324 15.3 2.2 G012324 19.1 3.5 G012289 14.3 1.8G012289 13.6 0.8 G012312 14.0 0.8 G012312 14.4 NA G012292 13.4 3.6G012292 12.9 3 G012330 13.3 1.9 G012330 12.9 0.6 G012315 13.0 0.1G012315 12.8 1.9 G012332 12.6 1.0 G012332 11.5 0.6 G012340 10.5 2.3G012340 13.9 1.8 G012301 9.2 0.1 G012301 NA NA G012336 7.5 1.6 G0123366.3 1.8 G012282 7.3 1.2 G012282 8.6 2.3 G012307 5.3 0.2 G012307 NA NAG012317 3.5 0.6 G012317 3.5 0.7 G012260 1.9 0.6 G012260 1.8 0.1 G0122541.6 0.2 G012254 0.7 0.3 G012266 1.0 0.1 G012266 1.9 0.6 G012256 0.8 0.4G012256 0.2 0 G012274 0.8 0.1 G012274 0.7 0.1 G012259 0.6 0.1 G0122590.6 0.1 G012262 0.5 0.1 G012262 0.6 0.2 G012276 0.5 0.0 G012276 0.8 NAG012257 0.4 0.1 G012257 0.1 0 G012277 0.4 0.1 G012277 0.4 0.1 G0122610.2 0.1 G012261 0.2 0.1 G012270 0.2 0.1 G012270 0.1 0 G012255 0.1 0.1G012255 0.1 0.1 G012258 0.1 0.0 G012258 0.1 0.1 G012263 0.1 0.0 G0122630.1 0 G012264 0.1 0.0 G012264 0.2 0.1 G012265 0.1 0.0 G012265 0.1 0G012267 0.1 0.0 G012267 0.2 0.1 G012268 0.1 0.0 G012268 0.1 0.1 G0122690.1 0.0 G012269 0.1 0.1 G012272 0.1 0.1 G012272 0.1 0 G012273 0.1 0.1G012273 0.1 0.1 G012275 0.1 0.0 G012275 0.2 0.1 G012278 0.1 0.0 G0122780.1 0.1 G012279 0.0 0.0 G012279 0.1 0.1 G012271 NA NA G012271 0.1 0*“selected dgRNA”, a subset of the tested guide RNAs

TABLE 8B KLKB1 editing data for sgRNAs delivered to primary cynomolgushepatocytes: Sets 1 & 2 SET 1 SET 2 Avg Std Dev Avg Std Dev GUIDE ID %Edit % Edit GUIDE ID % Edit % Edit G000644 35.9 3.5 G000644 37.4 2.3G000645 64.1 0.2 G000645 65 0.1 G009246 92.7 1.2 G009246 93 0.9 G00926784 1.6 G009267 83.3 1.9 G009285 82.5 0.3 G009285 80.7 0.7 G009321 46.817 G009321 46.3 12.8 G012102 91.4 0.5 G012102 92.4 0.1 G012144 45.3 0.6G012144 45.4 4.2 G012253* 92 0.3 G012253* 90.4 1.3 G012254 2.3 0.1G012254* 2.8 0.1 G012255 0 0 G012255 0.1 0 G012256 0.1 0 G012256 0.1 0G012257 0.1 0 G012257 0.2 0 G012258 NA NA G012258 0.1 0.1 G012259 0.30.1 G012259 0.3 0.1 G012260 0.6 0 G012260* 0.8 0.1 G012261 0.2 0.1G012261 0.2 0.1 G012262 0.3 0.2 G012262 0.4 0.1 G012263 0.2 0.1 G0122630.2 0.1 G012264 0.1 0 G012264 0.1 0 G012265 NA NA G012265 0.1 0 G0122660.3 0.1 G012266 0.3 0.1 G012267 0.2 0.1 G012267 0.1 0 G012268 0.1 0.1G012268 0.1 0 G012269 0.1 0 G012269 0.1 0 G012270 NA NA G012270* 4.8 0.7G012271 0.2 0.1 G012271 0 0 G012274 NA NA G012274 0.6 0.2 G012277 0.60.1 G012277 0.6 0 G012278 0 0 G012278 0.2 0.1 G012279 0.2 0.1 G0122790.1 0 G012280* 42.2 1.6 G012280* 39.1 5.6 G012282* 8.8 0.3 G012282* 10.3NA G012283* 50.5 NA G012283* 53.7 3.2 G012284 0.3 0.2 G012284 0.2 0G012285* 30.2 4.9 G012285* 30.5 0.8 G012287 0.1 0 G012287 0.1 0 G0122915.5 0.8 G012291* 4.8 1.3 G012292 6.4 0.3 G012292* 6.3 0.7 G012293* 86.60.1 G012293* 83.3 6.8 G012294* 53.6 1.5 G012294* 52.1 0.3 G012295* 6.70.1 G012295* 7 0.8 G012296* 51.4 2.1 G012296* 49 3.4 G012297 NA NAG012297* 41.9 0.4 G012299* 38.2 0.9 G012299 NA NA G012300* 28.5 4.6G012300 NA NA G012301* 7.6 2 G012301 NA NA G012302* 39.9 2.8 G012302 NANA G012303 13.8 1.1 G012303 NA NA G012304* 69.7 2.8 G012304 NA NAG012305* 16 0.1 G012305 NA NA G012307 0.3 0.2 G012307 NA NA G012308*57.7 3.9 G012308* 58.1 0.7 G012309* 55.9 7.3 G012309* 55.1 3.7 G012310*27 2.3 G012310* 27 1.9 G012311 4 0.1 G012311* 3.6 1.8 G012312* 14.4 0.5G012312* 14.7 0.3 *“selected sgRNA”, a subset of the tested guide RNAs

2.2. Screening of sgRNA in Primary Human Hepatocytes (PHH), Editing andProtein Knockdown

Three PHH lots (Hu8296, Hu8300, and Hu8284) were individually plated asdescribed in Example 1 and incubated at 37° C., 5% CO2 for 24 hoursprior to lipofection. A mixture of 6.88 μL of 10 μM sgRNA guide and 4.5μL of 500 ng/μl Cas9 mRNA was prepared in a total volume of 11.4 μlwater. A lipofection reagent as described in Example 1 was thawed toroom temperature. The guide/Cas9 mRNA mix was sequentially added with4.8 μL of 50 mM sodium citrate/200 mM NaCl (pH 5), 4.8 μL of lipofectionreagent, and 54 μL of molecular grade water, to prepare a total volume75 μL per sample. The lipofection sample was pre-incubated with media,William's E or Cellartis Power Primary HEP Medium (Takada, Cat. Y20020),containing 3% FBS or cynomolgus serum for 10 min at 37° C. prior toaddition to cells. Cells were transfected with 10 μL of preparedlipofection sample containing 300 ng of Cas9 mRNA and 302 ng guidesgRNA.

The cells were lysed 72 hours post-transfection for NGS analysis wasconducted as described in Example 1.

For cells to be utilized for secreted protein analysis by ELISA orintracellular protein analysis by western blotting, at 72 hourspost-transfection the media was aspirated and replaced with CellartisPower Primary HEP Medium (Takada, Cat. Y20020). Media was aspirated andreplaced every two days. For samples used to determine reduction ofsecreted protein, media was aspirated from wells and replaced with freshmedia which was incubated for 24-48 hrs prior to harvest. Media wascollected and transferred to 96-well PCR plates and stored at −20° C.prior to use in assays.

Table 8C and FIGS. 3A-3B show percent editing and secreted KLKB1 proteinlevels based on transfection of three PHH lots. Twenty guides werecompared in pairs of PHH lots, and determined to be highly correlated(Spearman R>0.8) as shown in FIGS. 3C-3E.

TABLE 8C KLKB1 indel frequency and secreted KLKB1 protein levels in PHHPHH Lot 1-HU8296 PHH Lot 2-HU8284 PHH Lot 3-HU8300 GUIDE Indel SecretedIndel Secreted Indel Secreted ID Freq SD KLKB1 SD Freq SD KLKB1 SD FreqSD KLKB1 SD G012253 0.32 0.01 1.54 0.03 0.63 0.06 0.24 0.00 0.49 0.010.43 0.11 G012254 0.12 0.01 7.22 0.33 0.40 0.09 0.57 0.05 0.15 0.00 1.610.06 G012255 0.02 0.00 15.72 0.70 0.22 0.02 5.05 0.26 0.03 0.01 5.330.08 G012256 0.10 0.03 9.26 0.13 0.43 0.05 1.57 0.31 0.11 0.00 2.69 0.07G012257 0.00 0.00 22.99 0.45 0.08 0.04 8.75 0.28 0.00 0.00 3.71 0.21G012258 0.13 0.01 4.24 0.02 0.26 0.02 3.89 0.03 0.09 0.02 2.76 0.00G012259 0.27 0.01 2.18 0.01 0.55 0.07 1.02 0.01 0.35 0.01 0.91 0.13G012260 0.69 0.03 0.07 0.02 0.73 0.03 0.05 0.01 0.82 0.03 −0.50 0.01G012261 0.06 0.00 14.26 0.77 0.38 0.03 3.39 0.11 0.07 0.02 3.84 0.28G012262 0.48 0.03 1.65 0.05 0.74 0.04 0.68 0.09 0.53 0.03 1.40 0.10G012263 0.42 0.02 2.48 0.06 0.67 0.03 0.78 0.02 0.53 0.03 0.55 0.16G012264 0.32 0.02 1.97 0.02 0.65 0.07 0.54 0.03 0.26 0.01 2.02 0.09G012265 0.11 0.00 6.24 0.02 0.29 0.09 1.36 0.10 0.11 0.01 1.10 0.03G012266 0.42 0.01 1.30 0.02 0.61 0.05 0.87 0.03 0.45 0.02 0.15 0.02G012267 0.72 0.00 0.23 0.02 0.79 0.02 0.14 0.00 0.82 0.00 −0.52 0.01G012268 0.20 0.01 4.78 0.14 0.44 0.01 1.65 0.06 0.30 0.01 1.05 0.03G012269 0.18 0.03 4.10 0.06 0.68 0.01 0.52 0.07 0.25 0.00 2.79 0.04G012270 0.48 0.01 0.46 0.02 0.77 0.02 0.21 0.00 0.67 0.06 −0.13 0.04G012271 0.10 0.02 3.16 0.02 0.58 0.06 0.79 0.04 ND 0.00 0.87 0.05G012272 0.12 0.02 6.42 0.09 0.53 0.06 1.16 0.03 0.21 0.01 0.09 0.02G012273 0.07 0.01 9.39 0.03 0.16 ND 0.82 0.04 0.07 0.00 2.14 0.06G012274 0.04 0.00 15.14 0.43 0.38 0.11 2.73 0.03 ND 0.00 4.13 0.04G012275 0.54 0.00 2.35 0.02 0.75 0.03 0.89 0.00 0.62 0.02 0.33 0.00G012276 0.01 0.00 20.54 0.24 0.09 0.00 4.56 0.18 0.03 0.00 3.92 0.03G012277 0.28 0.05 3.39 0.08 0.57 0.00 1.61 0.01 0.22 0.03 2.22 0.01G012278 0.22 0.01 1.61 0.03 0.64 0.04 0.50 0.00 0.42 0.02 −0.08 0.01G012279 0.40 0.05 2.56 0.08 0.59 0.03 1.51 0.02 0.53 0.03 −0.12 0.01G012280 0.39 0.02 1.34 0.06 0.55 0.04 0.52 0.01 0.44 0.02 0.08 0.00G012285 0.24 0.00 2.53 0.01 0.56 ND 1.23 0.06 0.26 0.00 1.60 0.06G012286 0.26 0.01 2.56 0.05 0.48 0.12 1.06 0.01 0.33 0.01 0.55 0.02G012290 0.13 0.00 14.44 0.06 0.60 0.01 5.45 0.06 0.29 0.00 2.41 0.04G012293 0.00 0.00 21.78 0.26 0.00 0.00 6.39 0.02 0.54 0.04 0.21 0.02G012294 0.23 0.01 2.12 0.16 0.62 0.02 0.39 0.06 0.34 0.02 0.29 0.08G012296 0.22 0.00 2.89 0.11 0.58 0.05 0.90 0.04 0.35 0.01 0.55 0.04G012298 0.37 0.01 2.19 0.01 0.68 0.05 0.72 0.03 0.63 0.01 −0.04 0.00G012303 0.40 0.03 0.52 0.02 0.70 0.05 0.38 0.00 0.38 0.03 0.04 0.00G012304 0.30 0.02 0.89 0.09 0.26 ND 0.20 0.01 0.27 0.03 1.31 0.06G012305 0.38 0.03 0.79 0.07 0.73 0.03 0.32 0.01 0.51 0.02 0.04 0.00G012308 0.18 0.02 1.79 0.13 0.72 0.03 0.53 0.04 0.33 0.01 −0.03 0.01G012319 0.26 0.00 1.45 0.03 0.25 0.02 3.62 0.76 0.32 0.03 0.80 0.00G012320 0.21 0.00 2.24 0.11 0.63 0.03 0.88 0.01 0.32 0.01 1.17 0.02G012321 0.45 0.00 0.49 0.07 0.72 0.01 0.49 0.09 0.60 0.07 −0.09 0.02G012323 0.28 0.00 1.32 0.01 0.48 0.12 0.75 0.17 0.33 0.00 0.01 0.02G012325 0.40 0.02 2.51 0.01 0.62 0.00 1.37 0.68 0.50 0.05 0.27 0.02G012334 0.10 0.00 7.52 0.18 0.19 ND 2.86 0.14 0.06 0.02 3.07 0.19G012335 0.27 0.02 3.78 0.26 0.47 0.10 1.42 0.09 0.42 0.01 0.11 0.02G009321 0.10 0.03 19.59 1.30 0.30 0.32 6.57 0.00 0.37 0.03 3.80 0.11(HOX9) Untreated 24.03 0.03 7.58 0.10 3.47 0.03

2.3. Screening of sgRNAs in PHH

Primary human hepatocytes (PHHs) were transfected with Cas9 mRNA andsgRNA as described in Example 1. The cells were lysed 72 hours posttransfection and NGS analysis was conducted as described in Example 1.

Percent editing was determined for sgRNAs comprising guide sequences ofTable 1 for two primer sets. The average percent editing for each guidein the two data sets is shown in Table 9A and FIGS. 4A-4B.

TABLE 9A KLKB1 editing data in primary human hepatocytes Set 1 Set 2 % %Guide ID Editing SD N Guide ID Editing SD N G013946* 49.9 2.2 2 G01394650.8 9.1 2 G000644 49.3 5.7 2 G000644 44.7 3.7 2 G013945* 48.6 1.9 2G013945* 50.3 1.6 2 G012102 48.1 4.4 2 G012102 45.8 0.3 2 G012320* 45 142 G012320* 41.9 15 2 G013938* 44.5 9.5 2 G013938 ND NA 2 G009285 42.10.7 2 G009285 41.4 4.9 2 G009246 40.5 0.6 2 G009246 39.1 3.1 2 G00050236.5 15 2 G000502 34.9 18 2 G000502 36.5 15 2 G000502 34.9 18.4 2G012304* 36.2 3.3 2 G012304 ND NA 2 G013886* 30.5 4 2 G013886* 37.2 0.82 G012325* 28.7 3.3 2 G012325* 33.9 0.9 2 G013896* 28.4 3.7 2 G013896*28.7 1.2 2 G009321 27.7 10 2 G009321 26.9 11 2 G012323* 27.4 7.2 2G012323* 30.6 6.5 2 G013925* 25.9 11 2 G013925* 28.7 12 2 G013922* 21 72 G013922* 19.3 3.3 2 G012327 20.5 2.3 2 G012327* 19.5 4.6 2 G01232218.6 3.8 2 G012322 13.9 4.5 2 G009267 18.3 1.9 2 G009267 20.4 4.8 2G013924* 17.4 9.3 2 G013924 ND NA 2 G013943* 14.2 9.5 2 G013943* 12.16.1 2 G000645 13.5 3.2 2 G000645 9.1 4 2 G013895* 13.4 3.1 2 G013895 146.6 2 G013941* 13 4.8 2 G013941 14.3 2.6 2 G013882* 10.4 8.1 2 G013882ND NA 2 G012329* 9.8 0.7 2 G012329* 8.9 0.9 2 G012300* 9.7 1.8 2 G012300ND NA 2 G013899 9.3 0.2 2 G013899* 7.1 4.4 2 G013874 9 2.3 2 G013874*9.5 3.6 2 G013931 8.9 1.6 2 G013931 ND NA 2 G012315 8.8 5.4 2 G012315 NDNA 2 G013902 6.2 3.9 2 G013902* 4.9 5.2 2 G013917 6.1 4.6 2 G013917* 7.16.5 2 G013916 5.6 0.6 2 G013916 4 0.8 2 G012324 5.2 2.9 2 G012324 4.83.9 2 G013912 5.1 2.9 2 G013912 4.6 1.1 2 G013913 4.8 0.4 2 G013913* 50.2 2 G013932 4.8 4.9 2 G013932 ND NA 2 G013900 4.6 2.1 2 G013900 4.60.7 2 G012340 3.3 1.1 2 G012340 2.2 1.7 2 G013873 2.8 0.8 2 G013873 3.61.1 2 G013884 2.3 1.1 2 G013884 3 0.3 2 G013903 1.4 0.4 2 G013903 1.30.5 2 G013901 1.2 1.3 2 G013901 1.4 1.3 2 G013889 0.9 0.2 2 G013889 1.11.2 2 G013930 0.9 1.1 2 G013930 1.3 0.1 2 G013893 0.8 0.3 2 G013893 1.80.4 2 G013919 0.8 0.2 2 G013919 0.6 0.4 2 G013891 0.7 0.1 2 G013891 0.30 2 G013934 0.6 0.2 2 G013934 0.9 1.1 2 G013894 0.3 0.4 2 G013894 0.60.8 2 G013906 0.3 0 2 G013906 0.7 0.3 2 G013914 0.3 0.1 2 G013914 ND NA2 G013928 0.3 0.2 2 G013928 0.5 NA 2 G013875 0.2 0.1 2 G013875 0.3 0.1 2G013878 0.2 0.1 2 G013878 0.3 0.1 2 G013880 0.2 0.1 2 G013880 0.1 0 2G013939 0.2 0.2 2 G013939 0.2 0.1 2 G013942 0.2 0.1 2 G013942 0.2 0.1 2G013870 0.1 0 2 G013870 0.1 0 2 G013871 0.1 0 2 G013871 0.1 0 2 G0138830.1 0 2 G013883 0.6 0.4 2 G013897 0.1 0 2 G013897 0.1 0 2 G013927 0.1 02 G013927 0 0 2 G013929 0.1 0 2 G013929 ND NA 2 G013935 0.1 0.1 2G013935 0.1 0 2 G013936 0.1 0 2 G013936 0.1 0 2 G013940 0.1 0 2 G0139400.1 0.1 2 G013921 ND ND 2 G013921 0.3 0.2 2 G013926 ND ND 2 G013926 0.20.1 2 *“selected sgRNA”, a subset of the tested guide RNAs

2.3.1 Screening of sgRNAs in PCH

Primary cynomolgus hepatocytes (PCHs) were transfected with Cas9 mRNAand sgRNA as described in Example 1 using increasing amounts of preparedlipofection sample to assay a dose responsive effect. The cells werelysed 72 post transfection and NGS analysis was conducted as describedin Example 1. Percent editing was determined for sgRNAs comprising guidesequences in Table 1 using two primer sets for amplification anddetection of indels. The average percent editing for each guide in thetwo data sets is shown in Table 9B and FIGS. 4C-4D.

The selected guide RNAs and corresponding editing data from Sets 1 and 2are marked with an asterisk (*) in Table 9B. When compared the datasetswere determined to be highly correlated (Spearman R=0.987).

TABLE 9B KLKB1 editing data in primary cynomolgus hepatocytes Set 1 Set2 Guide ID % Editing SD N % Editing SD N G013923 4.3 0 2 5 0.6 2 G0138771.5 0 2 1.3 0.2 2 G013884 0.4 0 2 0.2 0.1 2 G013929 0.3 0 2 0.5 0.1 2G013940 0.2 0 2 0.3 0.1 2 G013915 0.1 0 2 0.2 ND 2 G013939 9.3 0.1 210.1 0.3 2 G013870 4.8 0.1 2 4 1 2 G013879 3.4 0.1 2 3.1 0.6 2 G0123271.4 0.1 2 1.5 0.1 2 G013942 0.7 0.1 2 0.2 ND 2 G013883 0.3 0.1 2 0.2 0.12 G013926 0.1 0.1 2 0 0 2 G013887 0.9 0.2 2 0.7 0.7 2 G013930 3.1 0.3 23.2 1.3 2 G013902 0.9 0.3 2 0.7 0.4 2 G013903 0.7 0.3 2 0.9 0.1 2G013875 6.8 0.4 2 5.6 0.2 2 G013881 5.8 0.4 2 5.7 0 2 G013914 1.4 0.4 2ND ND 2 G013936 8.2 0.6 2 8.1 1.3 2 G013916 7.1 0.6 2 6.4 1.9 2 G0138746.9 0.6 2 6 0.4 2 G012340 3.6 0.6 2 3.7 0.9 2 G013880 1.9 0.6 2 1.9 0.12 G013919 0.9 0.6 2 0.7 0.4 2 G013878* 14.3 0.7 2 11.8 3 2 G013931 4 0.82 ND ND 2 G013872 11.1 0.9 2 11.7 1 2 G013909 4.8 0.9 2 5.3 0.5 2G013899 3 0.9 2 3 1.6 2 G013886* 15.4 1.1 2 15.2 0.4 2 G013944 2.1 1.1 22.3 0.5 2 G013876* 23.4 1.3 2 21.4 3.3 2 G013921 4.1 1.3 2 3.1 3.3 2G013913* 12.1 1.5 2 11.8 ND 2 G013871* 25.9 1.6 2 23.9 4 2 G013904 9 1.62 10.4 2.3 2 G013906 3.2 1.6 2 3.3 1.9 2 G013928 4.6 1.7 2 4.1 1.6 2G012324 3.9 1.8 2 4.4 2.4 2 G013910 3.9 1.8 2 4.8 1.8 2 G013892 2.7 1.82 2.8 1.8 2 G013943 3.1 2 2 3.7 2.3 2 G013920 2 2 2 2.9 1.4 2 G013889*29.4 2.1 2 31.4 0.5 2 G013938* 24.9 2.1 2 24.2 0.6 2 G012329* 12.4 2.1 211.4 0.5 2 G012315 4.1 2.1 2 ND ND 2 G013927 4 2.1 2 5.2 0.3 2 G0139182.3 2.1 2 2 1.6 2 G013935 4.1 2.2 2 4.3 1.1 2 G013941 7 2.3 2 8.7 1.3 2G012322 10.8 2.4 2 10.4 3.4 2 G013890 4.9 2.5 2 5.5 2.1 2 G013925 5.82.6 2 6.9 2.3 2 G012325* 11.8 2.8 2 11.6 3.4 2 G013888 6.7 2.8 2 7.1 1.62 G013897 11 3 2 11.9 1.6 2 G013900 6.6 3 2 7.7 4.2 2 G013917* 16.4 3.22 18.3 3.8 2 G013937* 30.4 3.8 2 26.5 6 2 G013932 5.6 4.4 2 ND ND 2G012304* 11.7 4.7 2 ND ND 2 G013895 6.8 4.7 2 6 3.2 2 G013896* 11.5 5 210.3 3.8 2 G013907* 36.1 5.5 2 36.2 5.7 2 G012320 8.9 5.7 2 9.2 4.6 2G013945* 19 6.6 2 20 6.1 2 G013924* 13.9 6.7 2 ND ND 2 G013882* 24.7 9 2ND ND 2 G013946* 27.2 ND 2 22.4 4.2 2 G013873* 23.6 ND 2 20.1 2.9 2G012323* 12.3 ND 2 11.8 1.5 2 G013934 8.3 ND 2 7.1 1.6 2 G013905 6 ND 24 3.3 2 G013898 5.7 ND 2 6 2.1 2 G013908 1.8 ND 2 3.6 1.3 2 G013911 1.8ND 2 3.4 1.6 2 G013894 0.3 ND 2 0.2 0.1 2 G013885 ND ND 2 5.9 0.2 2G013891 ND ND 2 3.3 0.7 2 G013893 ND ND 2 1.2 0.9 2 G013901 ND ND 2 42.24.2 2 G013912 ND ND 2 0.4 ND 2 G013922 ND ND 2 11.2 1.1 2 G013933 ND ND2 12.2 4 2 *“selected sgRNA”, a subset of the tested guide RNAs

Example 3: Dose Response Assays

3.1 Cross Screening of sgRNAs in PCH and PHH in 4-Point Dose ResponseAssays

Modified sgRNAs targeting human KLKB1 and the cynomolgus matched sgRNAsequences were tested in PHH and PCH in a dose response assay, using 16guides from the PHH guide screen described in Example 2.2. Lipofectionsamples including Cas9 mRNA and sgRNAs were prepared as described inExample 2.2. Primary human and cynomolgus hepatocytes were plated asdescribed in Example 1. Both cell lines were incubated at 37° C., 5% CO2for 48 hours prior to treatment with lipofection samples. Lipofectionsamples were incubated in Cellartis Power Primary HEP Medium (Takada,Cat. Y20020) containing 3% FBS at 37° C. for 10 minutes.

Post-incubation the lipofection samples were added to the human orcynomolgus hepatocytes in a 4-point dose response assay. The PHH werelysed 120 hours post-transfection and the PCH were lysed 168 hrspost-transfection and gDNAs were subjected to quantified PCR for NGSanalysis as described in Example 1.

The indel frequency of sgRNAs at concentrations 0.4 nM, 3.3 nM, 30 nM,and 90 nM in PHH cells is shown in Table 10 and FIGS. 5A-5B. SecretedKLKB1 protein levels of the sgRNAs determined by ELISA is shown in Table10 and FIGS. 5C-5D.

TABLE 10 KLKB1 editing data and secreted KLKB1 protein levels in PHHGuide concen- Avg Avg tration Indel secreted GUIDE ID (nM) Freq SD KLKB1SD G012253 90 0.54 0.04 5.84 0.29 30 0.64 0.05 4.06 0.09 3.3 0.31 0.0117.29 0.35 0.4 0.02 0.00 26.56 0.00 G012259 90 0.30 0.02 8.70 0.10 300.52 0.06 4.90 0.00 3.3 0.20 0.03 18.90 0.20 0.4 0.02 0.00 26.60 0.00G012260 90 0.73 0.04 2.20 0.00 30 0.84 0.00 0.30 0.30 3.3 0.71 0.02 4.000.20 0.4 0.20 0.01 25.10 0.30 G012267 90 0.80 0.03 2.10 0.10 30 0.890.01 0.30 0.00 3.3 0.76 0.05 4.00 0.20 0.4 0.29 0.03 20.70 0.20 G01227890 0.47 0.10 4.80 0.10 30 0.57 0.02 1.80 0.10 3.3 0.24 0.02 10.80 0.100.4 0.02 0.01 26.50 0.10 G012279 90 0.00 0.00 18.70 0.40 30 0.00 0.0021.10 0.30 3.3 0.00 0.00 26.60 0.00 0.4 0.00 0.00 26.60 0.00 G012280 900.42 0.08 6.90 0.10 30 0.69 0.01 4.70 0.10 3.3 0.21 0.00 23.30 0.10 0.40.01 0.00 26.60 0.00 G012293 90 0.48 0.05 5.10 0.10 30 0.70 0.00 4.200.00 3.3 0.32 0.02 15.20 0.60 0.4 0.03 0.00 26.60 0.00 G012294 90 0.390.08 7.37 0.08 30 0.48 0.04 6.44 0.06 3.3 0.13 0.01 22.57 0.25 0.4 0.010.00 23.49 0.73 G012298 90 0.48 0.14 9.30 0.60 30 0.52 0.00 6.20 0.103.3 0.20 0.07 21.50 0.50 0.4 0.01 0.00 25.50 0.50 G012303 90 0.68 0.056.00 0.00 30 0.75 0.04 3.80 0.30 3.3 0.43 0.03 15.80 0.20 0.4 0.02 0.0023.20 2.70 G012304 90 0.54 0.02 6.00 0.10 30 0.52 0.02 7.20 0.20 3.30.21 0.04 21.30 0.30 0.4 0.01 0.00 25.60 0.00 G012305 90 0.61 0.03 7.900.00 30 0.66 0.01 4.40 0.10 3.3 0.33 0.01 18.00 0.00 0.4 0.01 0.00 26.100.20 G012308 90 0.41 0.01 10.70 0.20 30 0.42 0.10 9.10 0.10 3.3 0.080.01 24.30 0.10 0.4 0.01 0.00 26.60 0.00 G012321 90 0.43 0.00 9.70 0.1030 0.61 0.13 5.20 0.00 3.3 0.17 0.01 21.70 0.20 0.4 0.02 0.02 26.50 0.10G012323 90 0.28 0.03 9.90 0.30 30 0.28 0.10 7.60 0.10 3.3 0.14 0.0322.40 0.30 0.4 0.01 0.00 26.60 0.00 untreated NA 22.61 0.21

The indel frequency of sgRNAs at concentrations 0.4 nM, 10 nM, 30 nM,and 90 nM in PCH cells is shown in Table 11 and FIGS. 5E-5F SecretedKLKB1 protein levels of the sgRNAs determined by ELISA is shown in Table11 and FIGS. 5G-5H.

TABLE 11 KLKB1 editing data and secreted KLKB1 protein levels in PCHGuide concen- Mean Mean tration Indel secreted guide (nM) Freq SD KLKB1SD G012253 90 0.25 0.01 0.82 0.01 30 0.38 0.05 0.77 0.00 10 2.76 0.090.44 0.07 0.4 13.26 0.32 0.01 0.00 G012259 90 1.13 0.02 0.70 0.01 300.81 0.12 0.69 0.01 10 3.95 0.41 0.37 0.04 0.4 12.88 0.96 0.00 0.00G012260 90 0.36 0.02 0.82 0.02 30 0.23 0.00 0.81 0.01 10 1.56 0.13 0.560.06 0.4 11.51 1.03 0.01 0.00 G012267 90 1.28 0.10 0.67 0.02 30 0.980.07 0.67 0.00 10 3.18 0.37 0.42 0.04 0.4 11.88 0.28 0.01 0.00 G01227890 1.01 0.06 0.61 0.03 30 0.82 0.01 0.63 0.02 10 3.16 0.19 0.30 0.05 0.412.08 1.14 0.00 0.00 G012279 90 9.88 1.25 0.00 0.00 30 9.44 1.07 0.000.00 10 12.49 0.14 0.00 0.00 0.4 12.12 0.25 0.00 0.00 G012280 90 4.320.20 0.44 0.02 30 3.08 0.18 0.46 0.00 10 7.70 0.21 0.13 0.02 0.4 11.691.13 0.00 0.00 G012293 90 0.80 0.22 0.80 0.03 30 0.54 0.09 0.80 0.02 102.56 0.16 0.59 0.00 0.4 11.79 0.23 0.01 0.00 G012294 90 6.96 0.48 0.390.09 30 6.21 0.29 0.41 0.03 10 15.19 1.13 0.12 0.00 0.4 24.96 3.85 0.000.00 G012298 90 18.40 0.36 ND ND 30 25.03 2.87 ND ND 10 24.81 2.89 ND ND0.4 23.59 3.91 ND ND G012303 90 18.34 4.33 0.16 0.06 30 16.05 4.43 0.170.00 10 26.04 3.42 0.03 0.00 0.4 27.82 2.74 0.00 0.00 G012304 90 5.751.35 0.52 0.04 30 6.14 0.68 0.54 0.01 10 17.23 1.75 0.21 0.04 0.4 24.900.95 0.00 0.00 G012305 90 19.27 4.43 0.07 0.02 30 18.57 3.56 0.11 0.0110 22.11 2.31 0.02 0.00 0.4 27.30 1.17 0.00 0.00 G012308 90 6.97 2.000.44 0.04 30 7.52 0.00 0.49 0.03 10 14.43 0.07 0.18 0.05 0.4 22.80 2.560.00 0.00 G012321 90 4.95 1.25 ND ND 30 5.96 0.07 ND ND 10 10.71 0.16 NDND 0.4 23.83 1.78 ND ND G012323 90 9.93 2.24 ND ND 30 8.97 3.13 ND ND 1015.75 0.20 ND ND 0.4 22.16 0.27 ND ND

Indel frequency and secreted KLKB1 protein levels were shown to beinversely correlated in both PHH and PCH as shown in FIGS. 5I-5J.

3.2 Cross Screening of sgRNAs in PHH and PCH in 7-Point Dose ResponseAssays

Lipid nanoparticle (LNP) formulations of sgRNAs targeting human KLKB1sgRNA sequences were tested in PHH and PCH in a dose response assay.

The LNPs were formulated as described in Example 1. The final LNPs werecharacterized to determine the encapsulation efficiency, polydispersityindex, and average particle size according to the analytical methodsprovided above.

Primary human and cynomolgus hepatocytes were plated as described inExample 1. Both cell lines were incubated at 37° C., with 5% CO2 for 24hours prior to treatment with LNPs. LNPs were incubated in mediacontaining 3% FBS at 37° C. for 10 minutes. Post-incubation the LNPswere added to the human or cynomolgus hepatocytes in a 7 point 3-folddose response curve. The cells were lysed 72 hours post-transfection andgDNAs were subjected to quantified PCR for NGS analysis as described inExample 1.

The indel frequency of sgRNAs at concentrations, 0.04 nM, 0.13 nM, 0.40nM, 1.19 nM, 3.58 nM, 10.75 nM, and 32.25 nM are shown in Table 12 andcorresponding dose response curves in FIGS. 6A-B for PHH and FIGS. 6C-Dfor PCH.

TABLE 12 Indel frequency and secreted KLKB1 protein for LNPs targetingKLKB1 in vitro PHH PCH Guide Mean Mean Mean Mean conc. Indel SecretedIndel Secreted GUIDE ID (nM) Freq SD KLKB1 SD Freq SD KLKB1 SD G01225332.25 0.91 0.00 −1.38 0.22 0.90 0.01 −1.58 0.10 10.75 0.89 0.00 −1.300.08 0.89 0.01 −1.57 0.07 3.58 0.89 0.02 −1.23 0.19 0.84 0.00 −1.39 0.071.19 0.83 0.01 −0.48 0.11 0.55 0.03 5.01 0.24 0.40 0.64 0.04 2.49 0.070.18 0.02 22.35 0.29 0.13 0.34 0.03 9.34 0.01 0.05 0.01 27.18 0.07 0.040.12 0.01 15.04 0.17 0.01 0.00 27.18 0.07 G012259 32.25 0.84 0.01 −1.120.21 0.95 0.01 −1.57 0.07 10.75 0.86 0.01 −1.17 0.11 0.93 0.01 −1.570.02 3.58 0.85 0.02 −1.00 0.06 0.80 0.00 −0.71 0.70 1.19 0.79 0.01 −0.280.04 0.44 0.04 8.93 0.23 0.40 0.63 0.02 2.37 0.19 0.12 0.03 25.94 0.050.13 0.36 0.03 8.32 0.31 0.01 0.00 27.18 0.07 0.04 0.13 0.01 12.97 0.410.00 0.00 27.18 0.07 G012260 32.25 0.93 0.00 −1.36 0.21 0.96 0.00 −1.320.18 10.75 0.92 0.01 −1.35 0.15 0.95 0.01 −1.41 0.37 3.58 0.92 0.01−1.43 0.19 0.87 0.02 −0.94 0.03 1.19 0.92 0.01 −1.37 0.27 0.50 0.00 7.330.02 0.40 0.90 0.00 −1.03 0.17 0.13 0.01 24.00 1.03 0.13 0.84 0.02 0.300.03 0.03 0.00 27.18 0.07 0.04 0.63 0.03 4.51 0.20 0.00 0.00 26.95 0.39G012267 32.25 0.94 0.03 −1.58 0.13 0.95 0.02 −1.88 0.02 10.75 0.94 0.01−1.50 0.08 0.94 0.02 −1.94 0.02 3.58 0.93 0.01 −1.57 0.13 0.84 0.00−1.48 0.10 1.19 0.91 0.05 −1.62 0.25 0.52 0.01 7.68 0.29 0.40 0.91 0.01−1.48 0.22 0.15 0.01 24.74 1.33 0.13 0.87 0.01 −0.77 0.07 0.04 0.0127.00 0.05 0.04 0.78 0.02 1.28 0.07 0.01 0.00 27.00 0.05 G012278 32.250.95 0.00 −1.63 0.18 0.95 0.01 −1.87 0.04 10.75 0.94 0.01 −1.64 0.100.94 0.01 −1.91 0.14 3.58 0.94 0.00 −1.59 0.05 0.82 0.02 −1.41 0.16 1.190.88 0.01 −1.38 0.20 0.44 0.01 8.23 0.17 0.40 0.65 0.03 0.29 0.14 0.080.03 26.13 1.19 0.13 0.34 0.02 5.68 0.20 0.02 0.00 27.00 0.05 0.04 0.120.01 12.54 0.43 0.01 0.00 27.00 0.05 G012279 32.25 0.00 0.00 −0.99 0.390.00 0.00 0.02 0.11 10.75 0.00 0.00 −0.56 0.05 0.00 0.00 8.49 0.34 3.580.00 0.00 3.54 0.08 0.00 0.00 26.50 0.03 1.19 0.00 0.00 12.69 0.56 0.000.00 27.00 0.05 0.40 0.00 0.00 15.91 0.26 0.00 0.00 26.85 0.17 0.13 0.000.00 17.60 0.18 0.00 0.00 27.00 0.05 0.04 0.00 0.00 17.32 0.00 0.00 0.0027.00 0.05 G012280 32.25 0.93 0.01 −1.68 0.27 0.87 0.01 −1.91 0.01 10.750.90 0.03 −1.56 0.20 0.83 0.01 −1.62 0.30 3.58 0.91 0.03 −1.55 0.11 0.560.02 2.88 0.53 1.19 0.85 0.01 −0.61 0.27 0.15 0.00 21.86 0.24 0.40 0.610.02 4.05 0.45 0.02 0.00 26.36 0.86 0.13 0.26 0.01 11.55 0.19 0.01 0.0027.00 0.05 0.04 0.08 0.00 15.47 0.38 0.00 0.00 27.00 0.05 G012293 32.250.91 0.00 −1.79 0.18 0.98 0.00 1.82 0.06 10.75 0.91 0.01 −1.63 0.19 0.960.02 −1.84 0.02 3.58 0.92 0.02 −1.65 0.27 0.90 0.00 −1.21 0.55 1.19 0.890.00 −1.27 0.55 0.54 0.02 3.40 0.13 0.40 0.82 0.02 −0.36 0.32 0.19 0.0222.12 0.61 0.13 0.60 0.00 3.31 0.19 0.04 0.02 26.91 0.09 0.04 0.32 0.0110.09 0.50 0.01 0.00 27.00 0.04 G012294 32.25 0.93 0.01 −1.58 0.28 0.900.02 −1.84 0.12 10.75 0.91 0.01 −1.41 0.14 0.89 0.00 −1.86 0.04 3.580.91 0.01 1.46 0.23 0.60 0.00 −0.15 0.19 1.19 0.82 0.02 −0.96 0.32 0.230.03 13.68 0.43 0.40 0.60 0.03 1.61 0.43 0.07 0.03 25.94 1.04 0.13 0.300.01 7.91 0.02 0.01 0.00 26.73 0.16 0.04 0.12 0.00 12.18 1.01 0.01 0.0027.00 0.05 G012298 32.25 0.95 0.00 −0.50 0.11 NA NA 15.77 0.15 10.750.95 0.00 −0.47 0.23 NA NA 27.00 0.05 3.58 0.91 0.00 −0.15 0.21 NA NA27.00 0.05 1.19 0.83 0.01 0.88 0.40 NA NA 27.00 0.05 0.40 0.59 0.01 6.540.52 NA NA 26.39 0.82 0.13 0.24 0.02 12.35 0.21 NA NA 27.00 0.05 0.040.08 0.00 16.29 1.12 NA NA 27.00 0.05 G012303 32.25 0.96 0.01 −1.27 0.140.88 0.01 −1.55 0.10 10.75 0.94 0.00 −1.39 0.18 0.83 0.02 −1.36 0.123.58 0.93 0.01 −1.28 0.15 0.40 0.02 8.20 0.71 1.19 0.90 0.01 −0.60 0.180.08 0.01 26.23 0.95 0.40 0.74 0.02 2.18 0.05 0.02 0.00 27.18 0.07 0.130.36 0.01 9.45 0.14 0.00 0.00 27.18 0.07 0.04 0.13 0.01 15.14 0.26 0.000.00 27.18 0.07 G012304 32.25 0.94 0.01 −1.37 0.32 0.96 0.00 −1.70 0.1910.75 0.95 0.01 −1.39 0.10 0.94 0.01 −1.72 0.08 3.58 0.91 0.00 −1.410.21 0.87 0.00 −1.52 0.01 1.19 0.84 0.02 −0.38 0.15 0.52 0.02 6.62 0.710.40 0.61 0.02 3.74 0.19 0.13 0.02 25.35 1.70 0.13 0.22 0.01 11.39 0.180.02 0.00 27.18 0.07 0.04 0.06 0.00 15.41 0.72 0.00 0.00 27.18 0.07G012305 32.25 0.95 0.01 −1.26 0.16 0.86 0.01 −1.53 0.11 10.75 0.94 0.00−1.37 0.13 0.75 0.02 −0.43 0.12 3.58 0.92 0.02 −1.30 0.15 0.33 0.0115.42 0.72 1.19 0.86 0.01 −0.26 0.07 0.06 0.00 27.18 0.07 0.40 0.65 0.033.85 0.04 0.01 0.00 27.18 0.07 0.13 0.27 0.04 10.94 0.24 0.00 0.00 27.180.07 0.04 0.08 0.01 15.12 0.54 0.00 0.00 27.18 0.07 G012308 32.25 0.950.01 −1.07 0.19 0.93 0.02 −1.66 0.12 10.75 0.95 0.01 −0.92 0.07 0.920.01 −1.44 0.02 3.58 0.88 0.02 −0.77 0.27 0.60 0.07 −0.18 0.32 1.19 0.750.01 1.00 0.10 0.34 0.03 14.44 0.04 0.40 0.31 0.05 7.89 0.27 0.08 0.0226.77 0.64 0.13 0.07 0.00 12.76 0.59 0.03 0.02 27.18 0.07 0.04 0.02 0.0114.75 0.43 0.00 0.00 27.17 0.05 G012321 32.25 0.93 0.00 −1.17 0.24 0.960.01 −1.68 0.19 10.75 0.93 0.01 −1.26 0.14 0.92 0.02 −1.60 0.12 3.580.88 0.02 −1.07 0.21 0.80 0.02 −0.84 0.10 1.19 0.85 0.00 −0.17 0.17 0.400.02 14.33 0.57 0.40 0.63 0.02 3.56 0.06 0.09 0.01 23.98 1.24 0.13 0.250.03 10.27 0.82 0.03 0.02 27.17 0.05 0.04 0.09 0.01 13.27 0.79 0.01 0.0027.18 0.07 G012323 32.25 0.89 0.01 −1.17 0.28 0.93 0.00 −1.74 0.12 10.750.90 0.00 −1.35 0.14 0.93 0.00 −1.72 0.03 3.58 0.84 0.01 −1.14 0.17 0.760.54 −0.36 0.32 1.19 0.73 0.01 0.20 0.14 0.35 0.01 15.43 0.17 0.40 0.490.01 4.28 0.04 0.07 0.01 25.63 1.00 0.13 0.23 0.02 10.12 1.03 0.01 0.0027.18 0.07 0.04 0.08 0.01 14.08 0.80 0.00 0.00 27.18 0.07 G013901 32.250.29 0.01 7.11 0.34 0.97 0.00 −1.58 0.48 10.75 0.29 0.00 8.89 0.40 0.980.00 −1.76 0.26 3.58 0.22 0.02 10.94 0.34 0.95 0.01 −1.81 0.19 1.19 0.150.02 14.44 0.70 0.79 0.01 −0.82 0.32 0.40 0.08 0.01 13.76 0.22 0.46 0.008.67 0.27 0.13 0.05 0.00 14.80 0.40 0.19 0.01 21.35 0.16 0.04 0.02 0.0014.97 0.59 0.05 0.00 26.84 0.19 untreated NA 0.00 0.00 13.62 0.95 0.000.00 25.77 2.09

3.3 Cross Screening of Lead sgRNAs in PCH and PHH in 7-Point DoseResponse Assays

Lipid nanoparticle (LNP) formulations of modified sgRNAs were tested inPHH and PCH in a dose response assay.

The LNPs described in Example 3.2 were used in this study.

Post-incubation, the LNPs were added to the human or cynomolgushepatocytes in a 7 point, 3-fold dose response curve. The cells werelysed 72 hours post-transfection and gDNAs were subjected to quantifiedPCR for NGS analysis as described in Example 1. For KLKB1 proteinanalysis the cells were lysed at day 8 post-transfection and whole cellextracts were subject to western blotting analysis as described inExample 1.

The indel frequency of sgRNAs at concentrations, 0.04 nM, 0.13 nM, 0.40nM, 1.19 nM, 3.58 nM, 10.75 nM, and 32.25 nM for PHH and PCH is shown inTable 13 and dose response curve data is illustrated in FIGS. 7A and 7B.Secreted KLKB1 protein levels of the sgRNAs determined by ELISA is shownin Table 13 and FIG. 7C for PHH and FIG. 7D for PCH.

TABLE 13 Indel frequency and secreted KLKB1 protein for LNPs targetingKLKB1 in vitro PHH PCH Guide Mean Mean Guide Mean Mean conc. Indelsecreted conc. Indel secreted GUIDE ID (nM) Freq SD KLKB1 SD (nM) FreqSD KLKB1 SD G012260 32.25 0.88 0.03 −0.07 0.04 32.25 0.97 0.01 −1.420.04 10.75 0.86 0.03 0.05 0.09 10.75 0.98 0.01 −1.32 0.03 3.58 0.82 0.080.03 0.04 3.58 0.95 0.00 −1.41 0.04 1.19 0.81 0.07 0.06 0.00 1.19 0.680.07 −0.08 0.03 0.40 0.84 0.00 0.14 0.00 0.40 0.26 0.04 14.46 1.24 0.130.79 0.04 0.55 0.01 0.13 0.06 0.01 24.37 0.74 0.04 0.73 0.02 1.80 0.010.04 0.01 0.00 25.32 0.79 G012267 32.25 0.88 0.05 0.11 0.02 32.25 0.980.00 −1.46 0.01 10.75 0.89 0.04 0.17 0.01 10.75 0.97 0.02 −1.28 0.143.58 0.87 0.05 0.16 0.05 3.58 0.94 0.00 −1.36 0.08 1.19 0.89 0.04 0.120.02 1.19 0.61 0.01 1.06 0.00 0.40 0.86 0.02 0.23 0.02 0.40 0.20 0.0315.86 0.74 0.13 0.87 0.02 0.54 0.00 0.13 0.05 0.00 25.73 1.85 0.04 0.800.03 1.54 0.06 0.04 0.02 0.00 26.39 0.16 G012293 32.25 0.85 0.00 −0.090.07 32.25 0.98 0.01 −1.43 0.02 10.75 0.76 0.15 −0.04 0.01 10.75 0.980.01 −1.32 0.06 3.58 0.78 0.02 0.06 0.08 3.58 0.98 0.00 −1.37 0.07 1.190.85 0.06 0.39 0.30 1.19 0.81 0.01 −0.89 0.04 0.40 0.74 0.08 0.71 0.010.40 0.35 0.03 9.93 0.49 0.13 0.71 0.02 2.52 0.12 0.13 0.09 0.00 23.260.06 0.04 0.45 0.03 7.55 0.09 0.04 0.02 0.00 25.17 0.05 G013901 32.250.31 0.07 9.02 0.05 32.25 0.98 0.00 −1.47 0.02 10.75 0.31 0.02 8.64 0.3010.75 0.98 0.00 −1.35 0.04 3.58 0.26 0.03 11.28 0.62 3.58 0.98 0.00−1.45 0.03 1.19 0.19 0.01 12.75 0.42 1.19 0.89 0.02 −1.32 0.09 0.40 0.130.02 15.06 0.73 0.40 0.58 0.05 2.65 0.28 0.13 0.06 0.01 15.61 1.11 0.130.23 0.05 15.10 0.22 0.04 0.04 0.00 16.27 0.46 0.04 0.08 0.00 23.21 0.82untreated NA 0.00 0.00 18.29 1.14 NA 0.00 0.00 25.96 1.35

For KLKB1 protein analysis, PHH were transfected with human KLKB1 guide,G012267, and lysed at day 8 post-transfection and whole cell extractswere subject to western blotting analysis as described in Example 1.Human KLKB1 protein levels across the 7-point dose response curve werecompared to untreated control and normalized to GAPDH is shown in FIG.7E.

Example 4—Off-Target Analysis of KLKB1 Guides

The biochemical method described in Example 1 was used to determinepotential off-target genomic sites cleaved by Cas9 targeting KLKB1.Guides selected based on results from experiments described above weretested for potential off-target genomic cleavage sites. Sixteen KLKB1targeting guides were evaluated for off-target genomic cleavage againstgenomic DNA from HEK293 cells at a 16 nM concentration (ATCC, Cat.#CRL-1573). KLKB1 guide G012267 and control guides with known off-targetprofiles were included in experiments (G000644 targeted to EMX1 forwhich 281 off-target sites have been detected, G00045 targeted to VEGFAfor which 6602 off-target sites have been detected); Frock et al., 2015,Tsai et al., 2015) were evaluated for off-target genomic cleavageagainst genomic DNA from pooled human PBMC at a 64 nM concentration. Thenumber of potential off-target sites detected in the biochemical assayusing genomic DNA from HEK 293 cells are shown in Table 14A. The numberof potential off-target sites detected in the biochemical assay usinggenomic DNA from PBMCs are shown in Table 14B. The percent of off-targetsites detected by the assay performed herein as compared to the numberof off-target sites noted in the literature for the EMX1 guide and theVEGFA guide are noted.

TABLE 14A Biochemical Off-Target Analysis with HEK293 cell genomic DNAGuide Off- Concentration target Guide ID Target (nM) sites G012253 KLKB116 122 G012259 KLKB1 16 77 G012260 KLKB1 16 153 G012267 KLKB1 16 136G012278 KLKB1 16 153 G012279 KLKB1 16 0 G012280 KLKB1 16 292 G012293KLKB1 16 126 G012294 KLKB1 16 132 G012298 KLKB1 16 155 G012303 KLKB1 1645 G012304 KLKB1 16 42 G012305 KLKB1 16 48 G012308 KLKB1 16 116 G012321KLKB1 16 107 G012323 KLKB1 16 14

TABLE 14B Biochemical Off-Target Analysis with human PBMC genomic DNAOff-Target sites Guide Target (percent*) G012267 KLKB1 61 G000644 EMX1242/281 (86%) G000645 VEGFA 4431/6602 (67%) *Percent is relative to theknown number of off-target sites for each of the guide target sites.

Example 5. Targeted Sequencing for Validating Potential Off-Target Sites

KLKB1 guides were selected based on experiments above for furtherevaluation. The targeted off-target approach described in Example 1 wasused to evaluate the target indel activity for the potential off-targetsassociated with these guides. The off-target sites tested in theexperiment were identified via the biochemical assay experimentsdescribed in Example 4 or in silico prediction as described in Example1.

In this experiment, 3 sgRNAs targeting human KLKB1 were evaluated. PHHwere cultured and transfected with LNPs comprising Cas9 mRNA and sgRNAof interest (e.g., a sgRNA having potential off-target sites forevaluation) as described in Example 1. Genomic DNA was isolated from thePHH and subjected to NGS and targeted off-target analysis as describedin Example 1.

The number of potential off-target sites evaluated in the assay and ofthose sites, off-targets that were successfully characterized by theassay followed by sites that were validated via manual inspection areshown in Table 15A.

TABLE 15 Targeted Off-Target Analysis Off-targets Off-targets ValidatedGuide ID evaluated characterized off-targets G012260 223 206 5 G012267181 171 1 G012293 360 347 4

Example 6. In Vivo Editing of the Humanized KLKB1 Locus in Hu KLKB1Mouse Model

Humanized mice that express human KLKB1 protein (Hu KLKB1 mouse model)were used in this study. The Hu KLKB1 mouse model comprises a humanizedKLKB1 locus in which the region from start codon to stop codon of mouseKLKB1 was replaced with the corresponding human genomic sequence.Animals were weighed and dosed at volumes specific to individual bodyweight. There were 5 groups total (N=4 with 2 male and 2 female mice).

LNPs containing modified sgRNAs (G12260, G12267, G12293, G12303, andG12321) and the Cas9 mRNA were dosed via the lateral tail vein at 0.3mg/kg based on total RNA cargo in a volume of 10 ml per kilogram bodyweight. The final LNPs were characterized to determine the encapsulationefficiency, polydispersity index, and average particle size according tothe analytical methods described in Example 1. At day 11 post-LNPadministration, mice were euthanized, and liver tissue was collected forDNA extraction. The tissues were lysed using a Zymo Research BashingBead Lysis Rack, and DNA was extracted using the Zymo Research DNAExtraction Kit according to the manufacturer's protocol. The extractedDNA was subject to PCR to be submitted for sequencing.

Blood was collected into serum separator tubes and allowed to clot for 2hours at room temperature followed by centrifugation. ELISA wasperformed on the serum aliquoted and diluted in a 96-well plate.

Editing observed in treated mice is shown in FIG. 8A and Table 16A.

TABLE 16A In vivo Editing Data in Hu KLKB1 Mouse Model % Editing GuideEditing SD N G12260 32.9 10.96 4 G12267 72.83 1.17 4 G12293 43.05 5.59 4G12303 14.38 5.60 4 G12321 35.53 11.11 4

Serum human KLKB1 protein levels, pre- and post-dose, were measuredusing the ELISA assay as described in Example 1. The results are shownin Table 16B and FIG. 8B.

TABLE 16B Secreted KLKB1 protein levels in Hu KLKB1 Mouse Model DoseGuide (mpk) Pre-dose 1 SD Post-dose SD N G12260 0.3 19.04 6.20 13.107.96 4 G12267 0.3 22.48 9.32 1.42 0.41 4 G12293 0.3 18.54 5.41 7.59 2.104 G12303 0.3 21.21 9.98 23.11 8.58 4 G12321 0.3 18.07 5.21 11.22 4.51 4

Serum human KLKB1 protein levels from the samples were measured usingthe electrochemiluminescence-based array (MSD) as described in Example 1and compared to baseline levels. The results are shown in Table 17 andFIG. 8C.

TABLE 17 KLKB1 protein level in vivo Dose Pre- Post- % Guide (mpk) doseSD dose SD serum KD N G12260 0.3 10.44 0.43 6.84 0.09 38 4 G12267 0.310.59 0.37 BLOD* —  97** 2 G12293 0.3 10.74 0.24 3.92 0.04 64 4 G123030.3 13.86 0.34 7.78 0.35  35** 4 G12321 0.3 8.38 0.1 3.92 0.16 55 4*Below limit of detection; **approximate

KLKB1 mRNA levels for each sequence were measured by quantitative PCR asdescribed in Example 1 and shown in Table 18 and FIG. 8D. Proteinreduction was confirmed by western blot analysis as described in Example1.

TABLE 18 qPCR results Fold Guide change SD N G12260 1.20 0.35 4 G122670.51 0.41 4 G12293 0.73 0.22 4 G12303 1.10 0.23 4 G12321 1.18 0.41 4 TSS1.01 0.17 2

Example 7. In Vivo Editing Activity of Human KLKB1 Guides in Hu KLKB1Mouse Model

Humanized KLKB1 mice described in Example 6 were used in this study andprepared using the same protocol. There were 5 groups total (N=5 with 2male and 3 female mice or vice versa). LNPs containing modified sgRNAsand mRNA encoding the Cas9 protein were dosed via the lateral tail veinat 0.3 mg/kg and characterized as described in Example 6.

At day 13 post-LNP administration, mice were euthanized. Liver tissueand blood was processed as described in Example 6 for sequencing andELISA analysis.

Table 19 and FIGS. 9A-9D show levels of KLKB1 editing, serumprekallikrein protein (detected using an ELISA that detects bothprekallikrein and kallikrein) (ug/ml), prekallikrein protein as percentof KLKB1 protein level in control TSS in treated mice, and thecorrelation of percent liver editing to percent prekallikrein protein,respectively. G012323 and G012253 guides were tested; however, editingwas not detected due to a failure of the NGS method.

TABLE 19 Percent Editing and Serum Prekallikrein of Certain Guides inHu-KLKB1 mouse model Serum Serum Dose % Prekallikrein Prekallikrein(mpk) Guide Sample Edit (ug/ml) (% TSS Mean) 0 TSS Mean 0.1 19.49 100Animal 1 0.1 14.77 76 Animal 2 0.1 18.29 94 Animal 3 0.1 14.82 76 Animal4 0.1 26.82 138 Animal 5 0.1 22.76 117 0.3 G012304 Mean 21.9 12.4 64Animal 1 24.7 9.53 49 Animal 2 21.0 9.88 51 Animal 3 29.3 7.94 41 Animal4 18.8 20.32 104 Animal 5 15.9 14.34 74 G012305 Mean 26.0 10.58 54Animal 1 20.6 9.09 47 Animal 2 32.5 8.85 45 Animal 3 27.9 8.13 42 Animal4 22.7 14.08 72 Animal 5 26.5 12.75 65 G012259 Mean 24.2 10.88 56 Animal1 41.0 5.83 30 Animal 2 20.3 9.96 51 Animal 3 27.6 7.42 38 Animal 4 9.417.83 91 Animal 5 22.8 13.36 69 G012278 Mean 24.0 10.91 56 Animal 1 29.87.15 37 Animal 2 35.2 6.99 36 Animal 3 28.6 6.16 32 Animal 4 18.2 17.2188 Animal 5 8.0 17.07 88 G012280 Mean 13.8 14.71 75 Animal 1 21.4 10.7055 Animal 2 24.0 8.92 46 Animal 3 4.8 20.46 105 Animal 4 8.9 17.34 89Animal 5 9.7 16.11 83 G012294 Mean 15.9 15.4 79 Animal 1 21.1 10.38 53Animal 2 15.9 9.69 50 Animal 3 15.2 15.37 79 Animal 4 14.9 19.13 98Animal 5 12.4 22.44 115 G012298 Mean 36.9 9.05 46 Animal 1 40.8 4.99 26Animal 2 46.6 5.26 27 Animal 3 44.9 5.38 28 Animal 4 25.3 15.01 77Animal 5 26.9 14.61 75

Example 8. In Vitro Dose Response of KLKB1 Gene Editing in Hu KLKB1Mouse Model

Humanized mice described in Example 6 were used in this study andprepared using the same protocol. There were 5 groups total (N=5 with 2male and 3 female mice or vice versa). LNPs containing G12267 and mRNAencoding the Cas9 protein were dosed at 0.3, 0.1, 0.03 and 0.01 mg perkg bodyweight and characterized as described in Example 6.

At day 13 post-LNP administration, mice were euthanized. Liver tissuewas processed as described in Example 6 for DNA sequencing. Blood wasprocessed as described in Example 6 and secreted human prekallikrein wasmeasured via an ELISA, which detects prekallikrein and kallikrein (also,called total kallikrein), as described in Example 1.

For RNA analysis, liver tissue was lysed using a Zymo Research BashingBead Lysis Rack, and RNA was extracted using the Qiagen RNeasy Mini Kit(Qiagen, Cat. 74106) according to the manufacturer's protocol. RNA wasquantified using a Nanodrop 8000 (ThermoFisher Scientific, Cat.ND-8000-GL). RNA samples were stored at −20° C. prior to use.

The SuperScript III Platinum One-Step qRT-PCR Kit (Invitrogen, Cat.11732-088) was used to create the PCR reactions. Quantitative PCR probestargeting Hu KLKB1 and internal control Ms PPIB were used in thereactions. The quantitative PCR assay was performed according to themanufacturer's specifications, scaled to the appropriate reactionvolume, as well as using the Hu KLKB1 and Ms PPIB probes specifiedabove. The StepOnePlus Real-Time PCR System (Thermo Fisher Scientific,Cat. 4376600) was used to perform the real-time PCR reaction andtranscript quantification according to the manufacturer's protocol.

Hu KLKB1 mRNA was quantified using a standard curve starting at 20 ng/uLof pooled mRNA from the vehicle control group, with five further 3-folddilutions ending at 0.06 ng/uL. Ct values were determined from theStepOnePlus Real-Time PCR System. Reduction of total secreted humanprekallikrein protein for cells treated with KLKB1 reagents wasdetermined by ELISA as described in Example 1.

Table 20 and FIG. 10 show percent editing, serum prekallikrein levels asa percent of TSS vehicle control treated mice, and mRNA transcriptlevels as a percent of TSS vehicle control treated animals.

TABLE 20 Percent Editing, KLKB1 mRNA (% of basal level) and SerumPrekallikrein Protein Levels (% of basal level) in Hu KLKB1 Mouse ModelDose % % TSS % TSS Guide (mpk) Editing protein mRNA SD TSS 0 Mean 0.1100 100.5 9.7 Animal 1 0.1 75.8 Animal 2 0.1 93.8 Animal 3 0.1 76.0Animal 4 0.1 137.6 Animal 5 0.1 116.8 G12267 0.01 Mean 3.9 91.9 100.19.5 Animal 1 4.4 55.3 Animal 2 3.8 57.3 Animal 3 4.5 126.2 Animal 4 4.2122.2 Animal 5 2.6 98.6 0.03 Mean 19.0 64.2 69.3 13.8 Animal 1 22.1 38.6Animal 2 0.3 51.0 Animal 3 26.9 78.9 Animal 4 21.3 80.5 Animal 5 24.372.2 0.1 Mean 55.4 23.3 48 11.4 Animal 1 52.1 17.6 Animal 2 52.7 19.6Animal 3 56.5 25.3 Animal 4 57.5 25.6 Animal 5 58.0 28.3 0.3 Mean 72.93.1 23 13 Animal 1 73.9 2.7 Animal 2 70.4 2.9 Animal 3 72.7 3.1 Animal 473.1 3.4 Animal 5 74.3 3.4

Example 9. Vascular Leakage Study

A study was performed to evaluate KLKB1 gene editing, total kallikreinprotein expression, and vascular leakage in humanized mice. Humanizedmice described in Example 1 were used in this study. There were 6 groups(N=5 with 2 male, 3 female mice per group). Animals were weighed anddosed at volumes specific to individual body weight.

LNPs containing a modified KLKB1 targeting sgRNA (G12267) and the Cas9mRNA were dosed via the lateral tail vein at 0.03 mg/kg, 0.1 mg/kg, or0.3 mg/kg based on total RNA cargo in a volume of 10 ml per kilogrambody weight or vehicle control (TSS).

At one day prior to the vascular leakage study, blood was collected andprocessed as described in Example 6, and secreted human prekallikreinwas measured via an ELISA, which detects prekallikrein and kallikrein(also, called total kallikrein), as described in Example 1.

The vascular leakage assay was performed as described in Example 1. Atnecropsy, liver tissue was collected and DNA extracted as described inExample 6 to measure KLKB1 editing. For dye quantification in thevascular leakage model, colon tissue was collected and processed asdescribed in Example 1.

The results for percent editing, serum hu KLKB1 protein levels, andvascular leakage are shown in Table 21 and FIGS. 11A-11B.

TABLE 21 Percent editing, KLKB1 protein levels, and vascular leakage inhuKLKB1 mice Serum Dose % Prekallikrein Colon (mg/kg) Editing (ug/ml)(OD) TSS-1 0 0.02 100.00 0.07 TSS-2 0 0.06 94.92 0.22 Control 0.3 0.192.99 0.30 G012267 0.03 16.48 82.97 0.26 G012267 0.1 40.46 48.97 0.20G012267 0.3 68.66 13.88 0.09

A separate study was conducted using similar methods to assess thepercent editing, serum prekallikrein levels, and vascular leakage fordurability over a 9-month period. Mice were dosed with modified KLKB1targeting sgRNA (G12267) and the Cas9 mRNA or a non-targeting sgRNA weredosed via the lateral tail vein at 0.1 mg/kg or 0.3 mg/kg based on totalRNA cargo in a volume of 10 ml per kilogram body weight. The durabilityof the dose response was observed where increased editing, decreasedprotein levels, and decreased vascular leakage levels were maintainedfor the length of the study.

Example 10. In Vivo Testing of KLKB1 Gene Editing in Non-Human Primates(NHPs)

In this example, a study was performed to evaluate KLKB1 gene editingand total kallikrein protein expression, and total kallikrein activitylevels in cynomolgus monkeys following administration of CRISPR/Cas9lipid nanoparticles (LNP) with mRNA for Cas9 protein and various guidesto the KLKB1 gene. Cynomolgus monkeys were treated in cohorts of n=3.This study was conducted with LNP formulations according to Example 1.Each LNP formulation contained a polyadenylated Cas9 mRNA (comprisingSEQ ID NO: 516) and gRNA (G013901, a cynomolgus specific KLKB1 guideRNA) with an mRNA:gRNA ratio of 2:1 by weight. Animals were dosed at1.5, 3, or 6 mg per kg doses based on total RNA cargo. Indel formation(percent editing) was measured by NGS. Total kallikrein activity andserum kallikrein protein level were measured as described in Example 1.

The study showed that knockout of KLKB1, which is part of a biologicalpathway that results in release of bradykinin, with G0013901 produced upto a 90% reduction in kallikrein activity in NHP groups, or more, arobust response that exceeds the target activity shown to achieve atherapeutically meaningful impact on HAE attack rates (60% kallikreinactivity reduction; Banerji, 2017). This study showed a dose-dependentcorrelation between increased editing rates, reduced plasma kallikreinlevels, and reduced kallikrein activity. The response has been durablethrough one year in NHPs. Circulating kallikrein protein and activitylevels are provided in Tables 22 and 23; and FIGS. 12A-12B.

TABLE 22 Kallikrein Activity (% of basal activity) TSS (n = 3) 1.5 mpk(n = 3) 3 mpk (n = 3) 6 mpk (n = 3) Day Mean SD Mean SD Mean SD Mean SD0 100 0 100 0 100 0 100 0 7 108.4 9 67.8 7 42.8 12.4 18.2 10.6 14 101.818.3 31.1 3.2 15.9 11 5.3 1.6 28 124.4 7.9 26.8 6.5 10.6 5.4 3.9 1.4 42117.8 5.8 19.8 11.9 8.6 4.4 3.6 0.4 56 130.3 32.5 8.8 5 3.4 0.6 4.2 2.270 109.2 15 6 1.7 3 0.4 3.9 1.6 84 121.3 25.7 10.6 3.8 4.2 2.1 4.7 2.5105 130.8 15.5 23.3 4.9 6 3.6 4.7 2.5 119 88.2 9.1 12.2 10.2 3.4 0.5 30.5 147 101.1 3.3 11.9 6.6 4.5 1.4 3 0.5 161 113.2 13.4 21 1.8 6 2.8 5.61.8 180 122.3 8.7 19.2 9.5 4.7 1 5 1.9 238 125.5 27.3 16.6 6.5 4.7 1.43.2 0.7 252 122.7 27 19.1 7.3 8.2 4.4 3.1 0.7 266 115 23.3 13.9 3.3 8.95.2 2.7 0.6 280 112.2 22.7 17.5 2.3 6.8 3.6 3 1 294 126.3 34.1 17 5.87.9 4.1 2.9 0.6 308 122.9 28.9 18.2 2.3 7.4 3.3 3 0.8 326 111.6 23 13.35.2 5.5 2.9 3.5 0.4 333 127.3 22.4 15.9 1.8 6.8 2.8 3.3 0.4 347 108.411.7 16.9 1 4.1 1.8 2.9 0.3 365 118 2.2 24.1 10.2 10 5.7 3.4 0.7

TABLE 23 Plasma Kallikrein Protein Levels (% of basal level) TSS (n = 3)1.5 mpk (n = 3) 3 mpk (n = 3) 6 mpk (n = 3) Day Mean SD Mean SD Mean SDMean SD 0 100 0 100 0 100 0 100 0 14 101.3 14.9 28.8 7.4 27.1 8.2 13.73.3 28 117.7 7 32.7 8.5 16.9 6.8 6.6 2.3 42 112.6 23.5 31.5 9.1 16 7 6.32.8 56 111 16 30.1 8.8 15.4 6 5.8 2.5 70 112.5 14.1 29.8 8 15.4 5.8 5.82.8 84 133.6 9 38.7 11.1 17.1 6.7 6.7 2.5 105 118.2 20.1 47 12.7 22.310.3 7.6 3.8 119 96 9.8 31.6 8.7 18.1 7.2 6.4 2.2 147 110.7 12 32.1 8.318.3 8.1 6.7 2.2 161 119.3 6.1 35.8 10.5 7.5 6.8 7.4 3.5 180 131 10.133.8 11.4 18 10.1 2.9 1 238 106.2 3.7 27.4 10.8 16.4 9.8 3.7 0.9 252114.3 21.4 27.9 9.1 15.6 7.1 3.9 0.8 266 117.6 8.7 28.7 9.7 21.8 10.43.7 0.5 280 93.4 22.1 33.7 7.1 24.6 9.8 6.3 2.9 294 122.1 23.7 30.4 13.824.6 14 7.4 4 308 93.2 12.8 20.6 13.8 27.6 14.2 6.2 3.1 326 N/A 29.7 7.323.8 12.5 8 2.8 333 130.2 25.5 30.9 6.4 25.3 12.4 7.8 2.3 347 106.9 1526 8.2 22.1 11.2 7.3 3.2 365 108.5 41.3 22.7 8 11.4 5.3 4.3 1.6

Tests of select NHP serum samples found no observed impact oncoagulation pathway biomarkers with KLKB1 knockout in NHPs at weeks 10or 15 (based on measuring prothrombin, APTT, and fibrinogen (all at week10), and Factor XII (at week 15)) when comparing TSS buffer controlgroups to treated groups.

The NHP study was repeated to evaluate KLKB1 total kallikrein proteinexpression, and total kallikrein activity levels in cynomolgus monkeysusing guide G012267 which includes a guide sequence fully complementaryto human KLKB1. The guide sequence of G012267 has one nucleotidedifference when compared to the G013901 which has a guide sequence fullycomplementary to cynomolgus KLKB1. The experimental protocol and LNPformulations in this study were essentially the same as described in theabove experiment, except animals (n=3) were only dosed at 3 mg per kgbased on total RNA cargo. Total kallikrein activity and serum kallikreinprotein levels were measured using the methods described in Example 1.

The study showed that knockdown of KLKB1 with G012267 produced up to a65% reduction in kallikrein activity in NHP groups. The response wasdurable through 9 months in NHPs. Circulating kallikrein protein andactivity levels are provided in Tables 24 and 25, and FIGS. 13A-13B.

TABLE 24 Kallikrein Activity (% of basal activity) TSS (n = 3) 3 mpk (n= 3) Day Mean Std. Dev Mean Std. Dev 0 100 N/A 100 N/A 7 90.9 13.7 68.711.3 15 87.7 6.6 51.3 18.7 28 90.5 2.5 39.3 24 42 96.5 4.8 31.9 19.5 5694.5 1.6 34.1 16.7 70 96.2 7.2 42.3 25.7 91 100.2 11.7 45.3 27.6 106100.2 6.3 45.7 14.3 120 99.8 7.4 46.5 11.1 134 115.2 8.5 47.5 32.4 14891.5 2 52.9 34.7 162 86.1 21.1 50.1 43.2 180 97.9 1.8 47.3 22.1 192 87.91.9 43 24.6 208 94.4 5.8 48 23 222 97 2.9 34.6 21.1 236 97.2 2.2 43.519.1 250 91.8 12 46.5 18.3 264 99.9 10.6 51.3 25.2 278 105.3 13.4 60.832.3

TABLE 25 Plasma Kallikrein Protein Levels (% of basal level) TSS (n = 3)3 mpk (n = 3) Day Mean Std. Dev Mean Std. Dev 0 100 N/A 100 N/A 7 99.65.1 72.3 13.3 15 89.7 13.1 50.6 9 28 93.9 5.2 52.8 22.3 42 90.3 8.6 52.725.5 56 104.2 17.3 49.9 19.1 70 93.9 5.2 52.8 22.3 91 90.3 8.6 52.7 25.5106 99.3 21.9 43.1 17.3 120 121.7 12.6 41.1 6.2 134 104.2 12 49.7 5.5148 93 15.8 47.5 11.8 162 100.3 15.7 56.2 26.7 180 106.8 10.9 44.1 33.2192 96.5 20.5 52 26.6 208 105.6 25 52.9 28.5 250 103.7 21.5 63.3 17.4264 101.4 7 67.3 15.4 278 94.3 14.4 62.6 16.7

1. A guide RNA comprising: a. a guide sequence at least 95%, 90%, or 85%identical to a sequence selected from SEQ ID NOs: 15, 8, and 41; b. aguide sequence comprising at least 17, 18, 19, or 20 contiguousnucleotides of a sequence selected from SEQ ID NOs: 15, 8, and 41; or c.a guide sequence selected from SEQ ID NOs: 15, 8, and
 41. 2. The guideRNA of claim 1, further comprising the nucleotide sequence of SEQ ID NO:202.
 3. The guide RNA of claim 1, wherein the guide RNA furthercomprises a nucleotide sequence selected from SEQ ID NO: 170, 171, 172,and 173 wherein the sequence of SEQ ID NO: 170, 171, 172, or 173 is 3′of the guide sequence.
 4. The guide RNA of claim 1, wherein the guideRNA further comprises a 3′ tail.
 5. The guide RNA of claim 1, whereinthe guide RNA comprises at least one modification.
 6. The guide RNA ofclaim 5, wherein the modification comprises (a) a 5′ end modification,(b) a 3′ end modification, or (c) a modification in a hairpin region. 7.(canceled)
 8. (canceled)
 9. The guide RNA of claim 1, wherein themodification comprises (a) a 2′-O-methyl (2′-O-Me) modified nucleotide,(b) a phosphorothioate (PS) bond between nucleotides, or (c) a 2′-fluor(2′F) modified nucleotide.
 10. (canceled)
 11. (canceled)
 12. The guideRNA of claim 1, further comprising the nucleotide sequence of SEQ ID NO:171 or
 173. 13. The guide RNA of claim 12, wherein the nucleotidesequence of SEO ID NO: 171 is modified according to the pattern ofnucleotide sequence of SEQ ID NO: 405: or wherein the nucleotidesequence of SEO ID NO: 173 is modified according to the pattern of SEOID NOs: 248-255 or
 450. 14. (canceled)
 15. (canceled)
 16. The guide RNAof claim 12, wherein the guide sequence comprises a sequence of SEQ IDNO: 15, 8, or
 41. 17. (canceled)
 18. (canceled)
 19. The guide RNA ofclaim 1, wherein the guide RNA is modified according to the pattern ofSEQ ID NO: 300, wherein the N's are collectively the guide sequence ofclaim
 1. 20. (canceled)
 21. The guide RNA of claim 19, wherein (a) theguide sequence is SEQ ID NO: 15 and the guide RNA is modified accordingto mG*mG*mA* UUGCGUAUGGGACACAAGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU; or (b) the guide sequence is SEO ID NO: 8 andthe guide RNA is modified according to mU*mA*mC*CCGGGAGUUGACUUUGGGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU: or (c) the guide sequence is SEO ID NO: 41 andthe guide RNA is modified according to mU*mA*mU*UAUCAAAUCACAUUACCGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU, wherein “mA,” “mC,” “mU,” or “mG” denote anucleotide that has been modified with 2′-O-Me, a * denotes aphosphorothioate bond, and N is a natural nucleotide.
 22. (canceled) 23.(canceled)
 24. A composition comprising a guide RNA of claim
 1. 25. Acomposition of claim 24, further comprising an RNA-guided DNA bindingagent or nucleic acid encoding an RNA-guided DNA binding agent.
 26. Thecomposition of claim 25, wherein the nucleic acid encoding an RNA-guidedDNA binding agent comprises an mRNA comprising an open reading frame(ORF) encoding an RNA guided DNA binding agent.
 27. The composition ofclaim 26, wherein the RNA-guided DNA binding agent is Cas9.
 28. Thecomposition of claim 27, wherein the Cas9 is S. pyogenes Cas9. 29.(canceled)
 30. (canceled)
 31. The composition of claim 24, wherein theguide RNA is associated with a lipid nanoparticle (LNP).
 32. Thecomposition of claim 31, wherein the LNP comprises an ionizable lipid.33. The composition of claim 32, wherein the ionizable lipid is(9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate, also called3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate.
 34. The composition of claim 31,wherein the LNP comprises(9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate, also called3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate, DSPC, cholesterol, and PEG2k-DMG.
 35. Apharmaceutical composition comprising a guide RNA of claim 1 and furthercomprising a pharmaceutical excipient or carrier.
 36. (canceled) 37.(canceled)
 38. (canceled)
 39. (canceled)
 40. (canceled)
 41. (canceled)42. (canceled)
 43. A method of (a) inducing a double stranded break or asingle stranded break within a KLKB1 gene in a cell; (b) reducingexpression of KLKB1 in a cell; (c) treating a subject having hereditaryangioedema (HAE); (d) treating or preventing angioedema associated withHAE, bradykinin production and accumulation, bradykinin-inducedswelling, angioedema obstruction of the airway, or asphyxiation: or (e)reducing total plasma kallikrein in a subject, the method comprisingcontacting a cell with a guide RNA of claim
 1. 44. The method of claim43, wherein the cell is in a subject.
 45. (canceled)
 46. The method ofclaim 43, wherein treating the subject comprises reducing the frequencyand/or severity of HAE attacks.
 47. (canceled)
 48. (canceled) 49.(canceled)
 50. (canceled)
 51. The composition of claim 27, wherein theORF encoding the Cas9 comprises an ORF from an mRNA of SEQ ID NOs:501-516.